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Diss Factsheets

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral feed: NOEL parental 400 mg/kg bw/day, NOEL 400 mg/kg bw/day, equivalent/similar to OECD 416, Heindel  et al. (1990).

Link to relevant study records
Reference
Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in line with GLP and the method was designed in line with good scientific principles. The study predates the inception of the OECD guideline 416 (2-generation reproductive toxicity), but is in basic compliance with the current standardised guideline. There is a good level of detail in reporting, sufficient to assess the quality of the data. The read-across approach has been used since phenoxyethanol is structurally similar to the test material but is more toxicologically active. The results presented are therefore taken to be the worst case scenario.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
The reproductive toxicity of the test material was investigated in Swiss CD-1 mice. Mice were exposed to the test material in diet at concentrations of 0, 0.25, 1.25 and 2.5% in diet equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1, 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).
GLP compliance:
yes
Limit test:
no
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: (P) 11 wks at the beginning of the continuous breeding test; (F1) 74 ± 10 days.
- Housing: Animals were housed by sex in solid bottom polypropylene or polycarbonate cages with stainless steel lids during the pre-mating period. The animals were then housed either individually or in breeding pairs.
- Diet: ad libitum.
- Water: Deionized water available ad libitum.
- Acclimation period: 2 weeks.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2 °C.
- Photoperiod (hrs dark / hrs light): 14 hours light (10 hours dark).
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Rate of preparation of diet: Weekly (diet was shown to lose approximately 5 % over 7 days).
- Mixing appropriate amounts with diet: Each dose level was blended into a small amount of ground diet, which was then added to a pre-weighed portion and mixed.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 98 days as breeding pairs in the continuous breeding phase, in the crossover trial and the offspring fertility assessment animals were cohabited for 7 days
- Proof of pregnancy: vaginal plug
- After successful mating each pregnant female was caged (how): In the offspring fertility test, animals were housed individually after the cohabitation period. During the continuous breeding phase, the animals cohabited for 98 days.
- Any other deviations from standard protocol: In the crossover mating trial, the parental animals from the continuous breeding phase were cross mated with control animals to determine the affected sex. Three groups of 20 pairs were used: control males x control female; high dose males x control females; control males x high dose females. In the offspring fertility check, the last litter produced from the continuous breeding phase and cohabited with a mate from a different litter within the same dose group.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No details of the methods used were reported. Aliquots of six representative samples were analysed during the study, the concentration was shown to be within 96 - 105% of the nominal concentration.
Duration of treatment / exposure:
- Range finding test: 14 days.
- Continuous breeding study: 7 day pre-mating dosing followed by 98 days cohabitation.
- In the cross-over mating trial parental animals selected from the continuous breeding study were necropsied after 3 weeks.
- The animals selected for the offspring fertility test were necropsied at the end of the test (after delivery).
Frequency of treatment:
Daily, dosed feed was available ad libitum, apart from the 7 days were breeding pairs were cohabited in the cross-over mating trial.
Details on study schedule:
- F1 parental animals not mated until 74 ± 10 days after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 21 days of age.
Remarks:
Doses / Concentrations:
4000, 2000, 400 and 0 mg/kg bw /day
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0, 0.25, 1.25 and 2.5%
Basis:
actual ingested
No. of animals per sex per dose:
- 8 animals/sex/group in each of the range finding dose levels
- 40 breeding pairs in the control group and 20 breeding pairs per dose were using in the continuous breeding phase of the study.
- In the crossover mating test 20 breeding pairs per group (3 in total) were used.
- In the offspring fertility test, 8 to 10 litters from the F1 generation from the control group and the 1.25 % dose level were selected for rearing to sexual maturity. At 74 ± 10 days one to three female and male pups were selected from each surviving litter and selected randomly for breeding. 19 breeding pairs per dose were used in this test.
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: A 14-day range finding study was included in order to select an appropriate dose for the study. Five dose groups and a control were exposed to 10, 7.5, 5.0, 2.5, 1.0 and 0.0 % in diet for two weeks. Animals were assessed for clinical signs, bodyweight changes and food consumption. Based on the results of this study, the appropriate doses were selected for the continuous breeding study. As both male and female animals lost 10 % of their bodyweight in the 7.5 and 10 % groups; 0, 0.25, 1.25 and 2.5 % were selected as the dosing levels in the continuous breeding trial.
Parental animals: Observations and examinations:
All animals (continuous breeding study, crossover mating trial and the offspring fertility test) were assessed for the following.

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes calculated as the average g/day/mouse
Sperm parameters (parental animals):
The following parameters examined in all male mice in all tests within the study; sperm count, sperm motility and sperm morphology.

In addition, the following were examined in the continous breeding test and the offspring fertility test (F0 and F1) animals; testis weight and epididymis weight.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring; number and sex of pups, stillbirths, live births, postnatal mortality, weight gain and fertility.

The number of litters produced by the F0 generation in the continuous breeding phase of the study was also evaluated.

GROSS EXAMINATION OF DEAD PUPS: no
Postmortem examinations (parental animals):
ORGAN WEIGHTS
In the male animals from the continuous breeding study, the females from the crossover mating test and both the males and females from the offspring fertility test the liver weights were recorded.

In addition the following organs and tissues from male animals in the continuous breeding test and the offspring fertility test were weighed: right epididymis, right testis, seminal vesicles and prostate.
Postmortem examinations (offspring):
ORGAN WEIGTHS
As listed above, the following organ weights from the pups produced in the continuous breeding study used in the offspring fertility test were examined: liver, right epididymis, right testis, seminal vesicles and prostate.
Statistics:
The findings from the continuous breeding study were assessed using the Cochran-Armitage test to determine whether there was a dose-related trend. In the cross-over mating trial, as it was not possible to test for a dose-related trend, the findings were assessed using a Chi squared test for homogeneity to determine the overall difference in fertility among groups. Fisher’s exact test was As a pairwise comparisons between the control and dosed groups.
Dose groups means for sex ratio and proportion of live pups were assessed using the Kruskal-Wallis test to examine overall difference and Jonckheere’s test to assess ordered differences. Pairwise comparisons of treatment group means were performed using the Wilcoxon-Mann-Whitney U test.

To prevent the number of pups per litter affecting the assessment of average pup weight an analysis of covariance was used, using the average litter size and the covariate including live and dead pups. Least-squares estimated of dose group means, adjusted for litter size were tested using an F test to assess overall equality and a t test to test for pairwise equality. To prevent interference from potential sex differences, these were performed with male and females separately and also both sexes combined.

An analysis of covariance was also used to adjust organ weights appropriately for bodyweight. Unadjusted body and organ weights were analysed by the Kruskal-Wallis and the Wilcoxon-Mann-Whitney U tests. Jonckheere’s test was used to test for dose-related trends.
Reproductive indices:
The following reproductive indices were calculated using equations below:

Mating index (%) = (Number of animals with copulatory plug/Number of animals cohabited breeding pairs) x 100

Fertility index (%) = (Number of fertile pairs/Number of animals with copulatory plug) x 100
Offspring viability indices:
The number of litters and live pups per litter were assessed per fertile breeding pair from this figure the treatment group means were determined. The proportion of live pups was defined as the number of pups born alive divided by the total number of pups produced by each breeding pair. The sex ratio was expressed as the proportion of male pups born alive out of the total number of live pups born to each fertile pair.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Three females died in the continuous breeding study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males only.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males only.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Test substance intake: No treatment related effects.
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
No treatment related effects.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
2.5 % EGPE in diet reduced the number of litters delivered per breeding pair and reduced litter size and number of live births.
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
The test substance was found to have a low general toxicity, with only three female dying in the continuous breeding test.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
In the continuous breeding study, only a minimal effect was noted on male bodyweight (2 % decrease) with no change on average in females. There was no significant effect on feed consumption. The approximate daily consumption was 5.6 g/day/mouse. At necropsy at the end of the cross-over mating test, there was a significant decrease in the bodyweight for males exposed to 2.5 % EGPE.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
There were no effects on feed consumption. The average daily test substance intake was approximately 400, 200 and 4000 mg/kg bw/day from the 0.25, 1.25 and 2.5 % groups respectively. The test substance intake varied in the females with the stage of gestation due to fluctuations in bodyweight.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
In the Cross-over mating test there were no differences between the treated and control males in respect to sperm concentration, percentage of motile sperm and percentage of abnormal sperm.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Continuous exposure in the continuous breeding test did not affect the number of breeding pairs able to produce at least one litter. Exposure to 2.5 % of EGPE was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the cross-over mating test, the results did not show which sex was affected by administration of the test material. The only difference was live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In the cross-over mating trial, when adjusted for bodyweight, the liver weight was found to be significantly increased in both males and females (21 and 60 % respectively). There were no significant difference in weight of the right testis, prostate and epididymis between controls and the 2.5 % EGPE males.
Dose descriptor:
NOAEL
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, liver weight and number of litters produced, live pup weight and litter size
Clinical signs:
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
High lethality was seen in the 2.5 % group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Dose related decrease in body weight
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No treatment related effects.
Gross pathological findings:
not examined
Histopathological findings:
not examined
VIABILITY (OFFSPRING)
Exposure to 2.5 % of EGPE in the continuous breeding test was found to reduce the number of litters delivered per breeding pair and significantly reduced the size and proportion of pups born alive. Further assessment of the litters demonstrated that in the high-dose group only 12 out of 20 pairs (60 %) had a fifth litter compared to 36 out of 40 (90 %) in the control. The number of pups per litter was not found to be affected. In the final litters produced by the continuous breeding test, pup lethality was pronounced during the lactation and post-weaning periods in the 1.25 and 2.5 %. By day 21 (weaning) only eight litters in the 1.25 and 2.5 % dosing groups had sufficient pups for the offspring fertility test. From birth to the start of mating, 12 of 87 (14 %) of the selected offspring for the fertility test in the control group, 20 of 113 pups (18 %) in the 0.25 % EGPE group, 33 out of the 84 pups (39 %) in the 1.25 % EGPE dose group and 66 out of the 76 pups (87 %) in the 2.5 % EGPE treatment group had died. Due to the high lethality in the 2.5 % group (25 of 32 males and 21 of 24 females), the offspring fertility test was performed with the pups from the 1.25 % dosing group.

BODY WEIGHT (OFFSPRING)
There was a significant dose related decrease in adjusted live pup weight during the continuous breeding test. In the cross-over mating test live pup weight was significantly decreased by 12 % in the control males crossed with the 2.5 % dosed females. The F1 pups selected for the offspring fertility test (the last litters produced by the continuous breeding test) were found to have dose-related decreased bodyweights at birth, weaning and at mating (the start of the offspring fertility test). At the end of the offspring fertility test, bodyweights were found to be decreased in both males and females dosed with 1.25 % EGPE.

SEXUAL MATURATION (OFFSPRING)
In the offspring fertility test, there were no statistically significant effects on the proportion of copulatory plug positive matings (mating index), fertile pairs (fertility index), pups born alive, or number of pups per litter when compared to the control pairs. Consistently, the live pup weights for the F2 generation were reduced. The offspring fertility test did not indicate any effects on sperm concentration, percentage of motile sperm or percentage of abnormal sperm.

ORGAN WEIGHTS (OFFSPRING)
The F1 pups used in the offspring fertility test were found to have increased liver weights when adjusted for bodyweight at the end of the test. There was no effect on right testis, prostate or epididymal weight. Seminal vesicle weight was found to be significantly decreased compared to controls.
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
400 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: body weight, liver weight and live pup weight
Reproductive effects observed:
not specified

Table 1: Reproductive performance

Parameter

Continuous Breeding Test

Crossover Mating Test

Offspring Fertility Test

Control

0.25% EGPE in diet

1.25% EGPE in diet

2.5% EGPE in diet

Control male x Control female

2.5 % treated male x control female

Control male x 2.5 % treated female

Control

1.25% EGPE in diet

No. with copulatory plugs/No. cohabited

 

18/18

18/20

14/17

18/19

19/19

Mating index (%)

100

90

82

95

100

No. fertile/No. with copulatory plugs

38/38

20/20

19/20

18/18

16/18

16/18

12/14

17/18

18/19

Fertility index (%)

88

88

86

94

95

Litters per breeding paira

4.8 ± 0.1

4.8 ± 0.1

5.0 ± 0.1

4.5 ± 0.2

Live pups per littera

11.0 ± 0.4

11.6 ± 0.5

11.3 ± 0.5

9.0 ± 0.8*

9.06 ± 1.01 (16)

9.75 ± 0.90 (16)

7.42 ± 1.03 (12)

11.1 ± 0.5 (19)

10.7 ± 0.4 (19)

Proportion of pups born alivea

0.98 ± 0.01

0.99 ± 0.01

0.98 ± 0.02

0.90 ± 0.60*

0.86 ± 0.09 (16)

0.96 ± 0.02 (16)

0.92 ± 0.06 (12)

0.98 ± 0.01 (19)

0.99 ± 0.01 (19)

Live pup weight (g)a

1.65 ± 0.02

1.59 ± 0.03

1.58 ± 0.02

1.52 ± 0.04*

Adjusted live pup weight (g)ab

1.65 ± 0.02

1.61 ± 0.02

1.59 ± 0.02*

1.48 ± 0.03*

1.71 ± 0.04 (14)

1.72 ± 0.04 (16)

1.51 ± 0.04 (12)*

1.55 ± 0.02 (19)

1.45 ± 0.02 (19)*

* Significantly different from control group (P < 0.05)

aMean ± SE. Number of observations included in parentheses were appropriate

bMeans adjusted for total number of live and dead pups per litter by analysis of covariance

 

Table 2: Bodyweights, organ weights and sperm parameters of male mice in the continuous breeding study and male mice in the offspring fertility test

Observationa,b

Continuous Breeding Study

Offspring Fertility Test

Control

2.5 % EGPE in feed

Control

1.25% EGPE in feed

Number of males

37

20

20

20

Bodyweight (g)

38.81 ± 0.59

36.63 ± 0.58*

34.61 ± 0.73

30.71 ± 0.59*

Organ weights

Liver (g)

2.05 ± 0.03

2.48 ± 0.05*

1.85 ± 0.04

2.05 ± 0.04*

Right epididymis (mg)

64 ± 4

54 ± 6

50 ± 1

43 ± 1

Right testis (mg)

135 ± 3

139 ± 5

124 ± 4

117 ± 4

Seminal vesicles (mg)

470 ± 15

446 ± 21

357 ± 15

306 ± 14*

Prostate (mg)

49 ± 3

45 ± 4

30 ± 4

33 ± 4

Sperm parameters

Sperm concentration (106sperm/g caudal tissue)

633 ± 34

668 ± 32

1054 ± 52

1041 ± 60

Motile sperm (%)

65 ± 4

69 ± 5

55 ± 3

60 ± 1

Abnormal sperm (%)

2.1 ± 0.2

2.6 ± 0.3

4.5 ± 0.4

5.9 ± 1.3

aMean ±SE

bOrgan weights adjusted for bodyweight by analysis of covariance

* Significantly different from control (P < 0.05)

 

Table 3: Bodyweights and liver weights of female mice from the cross mating study and the offspring fertility test.

Observationa,b

Cross Mating Study

Offspring Fertility Test

Control

2.5 % EGPE in feed

Control

1.25% EGPE in feed

Number of females

38

17

18

16

Bodyweight (g)

37.1 ± 0.07

36.1 ± 0.9

30.97 ± 0.52

28.91 ± 0.50*

Liver (g)

2.15 ± 0.07

3.44 ± 0.11*

1.81 ± 0.04

2.08 ± 0.05*

aMean ±SE

bOrgan weights adjusted for bodyweight by analysis of covariance

* Significantly different from control (P < 0.05)

 

Table 4: Bodyweights of the offspring from the continuous breeding test (litter 5) at birth, weaning and the beginning of the offspring fertility test

Age (days)

Continuous Breeding Test

Control

0.25% EGPE in diet

1.25% EGPE in diet

2.5% EGPE in diet

Birth (Day 0)a

Male

1.77 ± 0.03 (34)

1.70 ± 0.04 (15)

1.69 ± 0.07 (18)

1.60 ± 0.04 (12)*

Female

1.68 ± 0.03 (34)

1.66 ± 0.05 (15)

1.66 ± 0.07 (18)

1.52 ± 0.05 (12)*

Weaning (Day 21)b

Male

9.82 ± 0.44 (41)

9.43 ± 0.45 (42)

7.35 ± 0.27 (33)*

5.84 ± 0.22 (32)*

Female

9.80 ± 0.41 (39)

8.34 ± 0.32 (54)

7.56 ± 0.29 (32)*

5.57 ± 0.24 (24)*

Mating trial (Day 74 ± 10)c

Male

35.57 ± 0.87 (20)

34.77 ± 0.70 (20)

30.82 ± 0.62 (20)*

28.76 ± 1.94 (3)*

Female

28.00 ± 0.62 (20)

27.15 ± 0.60 (20)

25.69 ± 0.37 (20)*

24.24 ± 0.82 (3)*

* Significantly different from control group (P < 0.05)

aMean ± SE for male or female pup weight per litter; number of litters evaluated indicated in parentheses

bMean ± SE for individual pup weights; number of pups weighed indicated in parentheses

cMean ± SE for individual mice

Conclusions:
Under the conditions of the test, administration of the test material did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test material was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.
Executive summary:

The reproductive toxicity of the test material was investigated in Swiss CD-1 mice. Mice were exposed to the test material in diet at concentrations of 0, 0.25, 1.25 and 2.5% equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1.25 and 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating).

Under the conditions of the test, administration of the test material did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test material was found to be toxic to immature mice of both sexes. Under the conditions of the test, the NOAEL for both parental and reproductive toxicity was found to be 400 mg/kg bw/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
The quality of the database is high, the key study was performed in line with good scientific principles and reported in sufficient detail to address the endpoint. The lack of effects on fertility was confirmed in the supporting study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The key study, Heindel et al. (1990), studied the effects of 2-phenoxyethanol in Swiss CD-1 mice in a continuous breeding study. The study was performed in line with GLP according to the continuous breeding methodology which ensured that all stages of the reproductive cycle were encompassed in the test. Mice were exposed to the test material in diet at concentrations of 0, 0.25, 1.25 and 2.5% equivalent to 0, 0.4, 2.0 and 4 g/kg bw/day (a range finding study was conducted with concentrations of 0, 1.25 and 2.5, 5, 7.5 and 10% in feed for 2 weeks). Observations during the study included general toxicity, sperm analysis, reproductive performance, pup abnormalities and assessment of possible sex related effects (crossover mating). Under the conditions of the test, administration of the test material did not affect the ability to produce 5 consecutive litters in the continuous breeding phase of the study; there was a small decrease in the number of pups/litter and in pup weight in the high dose group, 2.5%. When examined in a crossover study, the affected reproductive component was considered to be from the female, however this was considered inconclusive after assessment, with the only finding being reduced pup weight. Fertility was only minimally compromised, however neonatal toxicity was observed, with increased lethality observed throughout lactation, weaning and puberty. Second generation fertility was found not to be affected, a small decrease in live pup weight was observed. The test material was found to be toxic to immature mice of both sexes. The study was reported to a high standard and was therefore assigned a reliability score of 2 in accordance with Klimisch (1997). Under the conditions of the test, the test material was found to have only minimal effects of fertility. A NOAEL of 400 mg/kg bw/day was concluded from the findings of the study.


 


The supporting study, Nagano et al. (1984), investigated the histopathological effects of 2-phenoxyethanol on the testes of male ICR mice. Male mice aged 6 weeks were dosed with the test material at 500 and 1000 mg/kg bw/day 5 days/week for 5 weeks via gastric intubation. Upon termination, testes were weighed and combined with the weight of the seminal vesicles and coagulating gland. Tissues were prepared for histopathological examination. Haematological changes were also investigated. The study was performed in line with good scientific standards, however, the results on 2-phenoxyethanol were reported in an assessment with several other chemicals using three different methods. The information presented on 2-phenoxyethanol was extremely brief, and therefore was assigned a reliability score of 3 in accordance with Klimisch (1997). Under the conditions of the test, the test material was found not to exhibit any effects on the testes of mice.


 


2-phenoxyethanol has been used as a read across substance since it is structurally similar to the substance to be registered. However it is anticipated to be more toxicologically active, and as such the results presented are taken to be the worst case scenario.


 


Justification for selection of Effect on fertility via oral route:


The continuous breeding study was selected as the key study as it was a thorough investigation on the effects of the fertility of the test material over multiple generations. The study was performed on a similar substance which was considered sufficiently similar to read-across the data to the registered substance in order to fulfil the data requirement.

Effects on developmental toxicity

Description of key information

Oral Route Key Study: Hamm (2022)


Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).


 


Oral Route Supporting Study: Hamm (2021)


Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.5 % (1 446.7 mg/kg/day).


 


Dermal Route WoE Read-Across Source: Palmer (1986)


Both maternal and embryo-foetal toxicity was observed at a dose of 1.40 mL/kg bw/day (1428 mg/kg bw/day). Given the physical chemical properties and ability to produce narcosis the embryo-foetal toxic effects were considered to be independent of the maternal toxicity, but likely to stem from the same physical-chemical mechanisms of toxicity. The 0.43 mL/kg bw/day (439 mg/kg bw/day) dose group was considered to be close to the threshold of maternal toxicity, but is  considered the NOAEL for maternal toxicity in this study. An enhancement of the incidence of reversible variations and anomalies were recorded in the individual foetuses, litter values however were not affected. No malformations were recorded at this dose level.


Under the conditions of the test, the NOAEL for maternal toxicity, embryo- and developmental toxicity was 0.43 mL/kg bw/day (439 mg/kg bw/day).


 


Dermal Route WoE Read-Across Source: Hoberman (1988)


Dosage dependent increasing scores for dermal irritation resulted from administration of all dosages of the test material as compared with control values. In addition, the 0.7 mL/kg/day dosage of the test material caused incidences of ptosis and/or urine-stained abdominal fur in the dams. On the basis of these findings, the neat test material was considered irritating to the skin of pregnant rats. Secondary effects of this irritation may have resulted in the observed altered fetal development.


Dosage dependent statistically significant observations of delays in fetal development (decreased average fetal body weight and delayed fetal ossification) resulted from administration of the test material at 0.14 mL/kg/day dosages and above.  There were no treatment related soft tissue effects in foetuses.  Significant increase in the incidence of foetuses with cervical ribs occurred for the 0.70 mL/kg/day group. All other foetal observations were considered to be reversible delays in ossification. Delayed ossification (sternum and/or pelvic) was noted in the all test material administered groups. Delayed pelvic ossification was found to be significant in all groups dosed with the test material, but neither alteration demonstrated a clear dosage dependent pattern.  No skeletal malformations were observed.


Under the conditions of the study, on the basis of no adverse fetal effects, and ptosis and/or urine-stained abdominal fur in dams developmental and maternal no-effect levels for the test material were 0.7 and ≤ 0.43 mL/kg/day respectively, equivalent to 710 and ≤ 439 mg/kg bw/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
A read across report will be added to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
0.43 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Dose descriptor:
NOAEL
Effect level:
0.43 other: mL/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 September 1984 to 14 October 1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed in compliance with GLP, and in basic compliance with OECD 414. The methods and results were well reported and sufficient to make a confident assessment of the accuracy of the conclusions and the suitability of the methods used. The study shows maternal toxicity, foetotoxicity and teratogenic effects at the high dose. The study is well written and demonstrates similar conclusions to other studies performed on this test material. The read-across approach has been used since phenylethyl alcohol is structurally similar to the test material but is more toxicologically active. The results presented are therefore taken to be the worst case scenario.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Doses of 1.40, 0.43 and 0.14 mL/kg/day were administered occlusively to the skin to Crl rats between days 6 to 15 of gestation. Maternal animals were observed for signs of toxicity and local irritation. On gestation day 20, the animals were sacrificed and developmental parameters were assessed in the pups in utero. In addition to the developmental toxicity study, an absorption study was included to give a brief picture on the behaviour of the substance when dosed dermally. For this purpose 3 animals from the low and high dose groups received a radiolabelled dose on the final day of treatment.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: CrL: COBS CD (SD) BR strain
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8-9 weeks
- Weight at study initiation: 170 - 242 g
- Housing: Individually caged in suspended galvanised metal cages with solid side and back, wire mesh front, floor and top.
- Diet: ad libitum
- Water: tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2 ºC
- Humidity (%): 53 ± 11%
- Air changes (per hr): 13 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light (0800 h to 2000 h)
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: Intrascapular region of each animal. A 7 x 5 cm area was clipped free of hair.
- Type of wrap if used: Occlusive, consisting of an aluminium foil patch held in place by a medical-type adhesive bandage.
- Time intervals for shavings or clippings: Animals were clipped on the day prior to treatment with electric clippers. Shaving was repeated as necessary.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The test site was not washed between applications.

TEST MATERIAL
- Amount(s) applied: The test substance was applied neat to the animals. The dose volumes for individual animals were calculated on day 6 of pregnancy and adjusted according to bodyweight on Days 8, 10, 12 and 14.

USE OF RESTRAINERS FOR PREVENTING INGESTION: no.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Animals were delivered time mated.
Duration of treatment / exposure:
Between gestation days 6 and 15.
Frequency of treatment:
Daily.
Duration of test:
Animals were sacrificed on gestation day 20.
Remarks:
Doses / Concentrations:
0, 0.14, 0.43 and 1.40 mL/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 143, 439 and 1428 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
Animals were received in two batches (designated A and B)
Group 1 (control) had a total of 25 animals (Batch A: 15; Batch B: 10)
Group 2 (143 mg/kg bw/day) had a total of 35 animals (Batch A: 15; Batch B: 20)
Group 3 (439 mg/kg bw/day) had a total of 25 animals (Batch A: 15; Batch B: 10)
Group 4 (1428 mg/kg bw/day) had a total of 35 animals (Batch A: 15; Batch B: 20)
Control animals:
yes
Details on study design:
- Dose selection rationale: The intermediate dose of the study was chosen as it was roughly equivalent to the highest dose used in the Mankes et al (1983) oral study.
Maternal examinations:
CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were handled daily to observe any symptoms of toxicity. Any local skin irritation was scored on each day of the dosing period.

BODY WEIGHT: Yes
- Time schedule for examinations: All rats were weighed on day 1 of gestation, then on days 3 and 6, then on alternate days thereafter until day 20.

FOOD CONSUMPTION
Food consumption was measured for the periods between each weighing.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20, animals were dissected and examined for congenital abnormalities and macroscopic pathological changes in maternal organs.
- Organs examined: The liver and kidneys of 13 high dose rats and 15 control rats were weighed and preserved for histological examinations.

HAEMATOLOGY
Blood samples were taken on gestation day 20 from 13 high dose rats and 15 control rats under light ether anaesthesia from the orbital sinus.
The following parameters were assessed: Packed cell volume, Haemoglobin, Red cell count, Mean corpuscular haemoglobin concentration, Mean corpuscular volume, Total white cell count, Platelet count, Differential WBC count (Neutrophile, lymphocytes, eosinophils, basophils and monocytes), Cell morphology, Thrombotest.

CLINICAL CHEMISTRY
The following parameters were assessed: Total protein, Albumin, Globulin, Urea nitrogen, Creatinine, Sodium, Potassium, Calcium, Inorganic phosphorous, Chloride, Cholesterol, Glucose, Alkaline phosphatase, Glutamic-pyruvic transaminase, Glutamic-oxaloacetic transaminase, Bilirubin.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes, all per litter
- Soft tissue examinations: Yes, half per litter
- Skeletal examinations: Yes, half per litter
Statistics:
Group mean values were calculated for litter size, embryonic deaths and pre- and post-implantation loss. Two methods of calculation were used, (A and B), the first (A) included all surviving animals that provided evidence of pregnancy (including those that had total resorption) and the second (B) included all animals with live young at termination.

More weight was put to Mean B when group sizes were low to prevent animals exhibiting total resorption skewing the results unrealistically. Mean A was considered more appropriate when several animals exhibited total resorption.

Mean B only was calculated for litter weights and mean foetal weights.

Statistical analysis of incidences of abnormalities was considered unnecessary.

Statistical analyses were performed on litter data, using the litter as the basic sample unit and non-parametric tests (Jonckheere and Kruskal-Wallis) as these values are not considered to follow a normal distribution. Analysis of covariance followed by Williams' test was used to assess intergroup differences in mean organ weights. The Kruskal-Wallis test was also used to analyse intergroup differences in the results of the biochemical and haematological examinations.
Indices:
Abnormalities were assessed as two categories.
Selected changes: Structural changes occurring at a high incidence at the high dosage and with an expected control frequency of 2 or less.
Other changes: Structural changes with a higher expected control incidence, or which did not show a marked increase at the highest dosage.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
1428 mg/kg bw/day group
From the pilot study, it was found that the test material was absorbed rapidly and extensively.
3/35 animals either died or were sacrificed during the course of the study due to progressively marked clinical signs of toxicity. At this dose level suppression of mean food intake and growth rate was noted in the maternal animals.

439 mg/kg bw/day group
It was hypothesised that this dose was close to the threshold for maternal toxicity as only one female in this group demonstrated the same clinical signs noted in the highest dose group. All other animals in this group were asymptomatic and there was no clear evidence of maternal toxicity when compared to the control group.

143 mg/kg bw/day group
No maternal toxicity was noted at this dose level.

Two animals in the top dose group were found dead on day 13 of gestation. One had already been sacrificed on day 12 due to poor condition. All three animals exhibited typical signs for this dose group. The animal sacrificed on day 12 also had in addition rapid respiration, blood in the urine and appeared to be unable to walk. At autopsy, the only macroscopic finding was a slight congestion of the dorsal subcutis in one of the animals.
A brownish deposit became apparent on the dosing site in all animals in the high dose group towards the end of the dosing period. This was found to be moist on removal of the bandage, but soon dried forming a crust.
With the exception on a single non-pregnant animal walking on its toes on day 16 of the study, there were no over signs of reaction to treatment at the two lower doses.
The majority of clinical signs associated with dosing were observed in the highest dosing group which developed at around day 12 of gestation. Most animals in this group showed irritability, hunched posture, walking on toes, pilo-erection and peri-orbital staining, some animals were completely asymptomatic. Less frequently signs such as suspected blood on the under cage tray paper, slight oedema and perineal staining were also noted.
Dosing of one animal was discontinued from day 12 of gestation due to severe irritability and hunched and emaciated appearance.
At the end of treatment, marked recovery was noted in all animals, by termination only one animal still exhibited peri-orbital staining, however the brown crust was flaking off revealing normal coat regrowth.
No effect on food consumption was noted in the lower two dosing groups. The highest dose group was suppressed during the later part of the dosing period, however at the end of treatment this did return to the control levels.
Upon initiation of dosing, a slight reduction in body weight was noted in all groups including controls, however all groups showed overall weight gain during the study.
No difference in body weight was noted between the control and the two lower dosing groups. However in the highest dosing group, bodyweight gain was markedly retarded compared to controls, which progressed through the continuing treatment. This was noted for all animals in this group and was not exclusive to the animals that resorbed their litter. Although recovery was noted after dosing was complete, the bodyweight of these animals at termination was depressed.
Apart from lower alkaline phosphatise levels, no other differences of great magnitude were recorded. Statistically significant lower values for creatinine and glutamic-pyruvic transaminase and statistically significant higher values for white cell count, these changes were marginal but significant.
There were no differences in organ weights adjusted for bodyweights in treated animals.
Mean ovulation rate and pre-implantation loss was comparable to controls. A higher mean implantation rate was observed. The overall pregnancy rate was similar for all the groups.
Although the animals that died during the study showed marked clinical signs, at autopsy, there were no findings that were considered to have attributed to the death of the animal.
Dose descriptor:
NOAEL
Effect level:
0.43 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
1428 mg/kg bw/day group
Total loss (resorption) of 5/23 litters and 50 % or more embryo-foetal wastage in a further 4/18 litters was observed at this dose level. Death generally occurred early in pregnancy. Along with the lower litter sizes, depressed foetal weight was also noted at this dosing level.

There were virtually no morphologically normal foetuses in this dose group. A wide range of soft and skeletal changes were observed. Certain characteristic predominated indicative of certain syndromes, but there was also a high incidence of non specific changes in this dose group. Incomplete ossification was also observed at this dose.

In more than 40 % of foetuses (70 % of the litters) examined, anopthalmia/microphthalmia, ventricular septal defects, defects/irregularities affecting thoracic, lumbar and sacrocaudal vertebrae the latter associated with short and/or kinky tail, defects of the thoracic ribs and occurrence of cervical ribs.

439 mg/kg bw/day group
Litter parameters in this group were comparable to the control. The specific changes noted in the highest dose group were occasionally found in foetus of this group. The number of foetuses with reversible variations, delated ossification or anomalies (cervical rib(s), thoracic changes and moderate degrees of reduced ossification) was found to be enhanced above control levels (which were not zero) The incidence of malformations was not increased over control levels.

143 mg/kg bw/day group
Litter parameters in this group were unaffected. Structural changes were found to be equivocal in this group. When assessed individually, the incident and distribution of changes could not be attributed to treatment.

All other miscellaneous types of skeletal change and soft tissue examinations allocated to skeletal staining, the increases incidence of litters and foetuses affected occurred only in the highest dose group.
Dose descriptor:
NOAEL
Effect level:
0.43 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Concentration Conversions

Concentrations in mL/kg bw/day were converted to mg/kg bw/day based on a density of 1.02 g/cm³ for phenylethyl alcohol. Therefore the test concentrations of 0, 0.14, 0.43 and 1.40 mL/kg bw/day are equivalent to 0, 143, 439 and 1428 mg/kg bw/day.

Table 1: Incidence of visceral changes in foetuses allocated to visceral examination

Observation

Litters

Foetuses

Number (Percent)

Number (Percent)

Group

 0 mg/kg bw/day

 143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

 0 mg/kg bw/day

 143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

Examined

21

30

22

18

115

178

132

82

Affected

Selected effects

0

4 (13.3)

4 (18.2)

18 (100)

-

8 (4.5)

5 (3.8)

81 (98.8)

Other effects

4 (19.0)

7 (23.3)

7 (31.8)

11 (61.1)

7 (6.1)

8 (4.5)

14 (10.6)

16 (19.5)

Selected Effects

Anopthalmia/microphthalmia

-

1 (3.3)

-

13 (72.2)

-

1 (0.6)

-

34 (41.5)

Lenticular lesion/retinal fold

-

-

-

13 (72.2)

-

-

-

24 (29.3)

Arterial defect

-

-

-

9 (50.0)

-

-

-

18 (22.0)

Ventricular septal defect

-

-

1 (4.5)

18 (100)

-

-

1 (0.8)

71 (86.6)

Venous irregularity

-

-

1 (4.5)

6 (33.3)

-

-

1 (0.8)

6 (7.3)

Caudal irregularity

-

-

-

18 (100)

-

-

-

52 (63.4)

Digital defect

-

-

-

9 (50.0)

-

-

-

15 (18.3)

Subcutaneous oedema

-

-

-

6 (33.3)

-

-

-

7 (8.5)

Atelectasis

-

-

-

5 (27.8)

-

-

-

6 (7.3)

Dilated brain ventricles

-

1 (3.3)

-

7 (38.9)

-

1 (0.6)

-

11 (13.4)

Displaced/enlarged adrenal

-

-

1 (4.5)

7 (38.9)

-

-

1 (0.8)

9 (11.0)

Dilated renal pelvis/ureter

-

3 (10.0)

2 (9.1)

8 (44.4)

-

6 (3.8)

2 (1.5)

14 (17.1)

Other Effects

Displaced testis (es)*

2 (9.5)

3 (10.0)

2 (9.5)

6 (40.0)

3 (4.9)

3 (3.2)

2 (3.0)

6 (15.8)

Haemorrhage(s)

2 (9.5)

4 (13.3)

5 (22.7)

6 (33.3)

3 (2.6)

5 (2.8)

11 (8.3)

6 (7.3)

Absent innominate artery

2 (9.5)

-

-

-

2 (1.7)

-

-

-

Absent/irregular lung lobes

-

-

-

3 (16.7)

-

-

-

3 (3.7)

Diaphtagmatic hernia

-

-

1 (4.5)

1 (5.6)

-

-

1 (0.8)

1 (1.2)

Reduced thymus

-

-

-

2 (11.1)

-

-

-

3 (3.7)

Absent/reduced kidney

-

-

-

2 (11.1)

-

-

-

2 (2.4)

Reduced urinary bladder

-

-

-

2 (11.1)

-

-

-

2 (2.4)

Anal atresia

-

-

-

1 (5.6)

-

-

-

1 (1.2)

Displaced oesophagus

-

-

-

1 (5.6)

-

-

-

1 (1.2)

Displaced ureters

-

-

-

1 (5.6)

-

-

-

1 (1.2)

Displaced thoracic viscera

-

-

-

1 (5.6)

-

-

-

1 (1.2)

Situs inversus

-

-

1 (4.5)

-

-

-

1 (0.8)

-

Malrotated limbs

-

-

-

2 (11.1)

-

-

-

2 (2.4)

Litters and foetuses are counted only once in the totals affected but they may occur in more than one category

*Values calculated on the basis of the number of males and number of litters containing males

 

Table 2: Incidence of visceral changes in foetuses allocated to skeletal examination

Observation

Litters

Foetuses

Number (Percent)

Number (Percent)

Group

 0 mg/kg bw/day

143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

 0 mg/kg bw/day

 143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

Examined

21

30

22

18

118

178

129

79

Affected

Selected effects

1 (4.8)

1 (3.3)

-

7 (38.9)

1 (0.8)

1 (0.6)

-

14 (17.7)

Other effects

3 (14.3)

2 (6.7)

3 (13.6)

13 (72.2)

3 (2.5)

2 (1.1)

3 (2.3)

27 (34.1)

Selected Effects

Anopthalmia/microphthalmia

1 (4.8)

1 (3.3)

-

5 (27.8)

1 (0.8)

1 (0.6)

-

12 (15.2)

Hydrocephaly

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Subcutaneous oedema

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Other Effects

Displaced testis (es)*

2 (9.5)

1 (3.7)

-

6 (40.0)

2 (3.3)

1 (1.1)

-

8 (20.0)

Haemorrhage(s)

-

1 (3.3)

-

-

-

1 (0.6)

1 (0.8)

-

Absent/irregular lung lobes

-

-

1 (4.5)

-

-

-

-

-

Displaced kidney

-

-

-

8 (44.4)

-

-

-

9 (11.4)

Displaced uterus

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Situs inversus

-

-

2 (9.1)

-

-

-

2 (1.6)

-

Exophthalmas/ablepharia

1 (4.8)

-

-

-

1 (0.8)

-

-

-

Umbilical protrusion

-

-

-

6 (33.3)

-

-

-

6 (7.6)

Malrotated/flexed limbs

-

-

1 (4.5)

2 (11.1)

-

-

1 (0.8)

3 (3.8)

Litters and foetuses are counted only once in the totals affected but they may occur in more than one category

*Values calculated on the basis of the number of males and number of litters containing males

Table 3: Incidence of skeletal changes in foetuses allocated to skeletal examination

Observation

Litters

Foetuses

Number (Percent)

Number (Percent)

Group

 0 mg/kg bw/day

 143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

 0 mg/kg bw/day

 143 mg/kg bw/day

 439 mg/kg bw/day

 1428 mg/kg bw/day

Examined

21

30

22

18

118

178

129

79

Affected

Selected effects

6 (28.6)

10 (33.3)

17 (77.3)

18 (100.0)

7 (5.9)

13 (7.3)

38 (29.5)

79 (100.0)

Other effects

1 (4.8)

1 (3.3)

1 (4.5)

4 (22.2)

1 (0.8)

1 (0.6)

1 (0.8)

4 (5.1)

Selected Changes

Cervical rib(s)

1 (4.8)

5 (16.7)

14 (63.6)

18 (100.0)

1 (0.8)

6 (3.4)

30 (23.3)

74 (93.7)

Extra thoracic rib(s)

3 (14.3)

1 (3.3)

4 (18.2)

10 (55.6)

3 (2.5)

1 (0.6)

5 (3.9)

27 (34.2)

Displaced pelvic attachment

1 (4.8)

-

1 (4.5)

9 (50.0)

1 (0.8)

-

1 (0.8)

26 (32.9)

Defects/irregularities of:

Thoracic vertebrae

2 (9.5)

3 (10.0)

5 (22.7)

17 (94.4)

2 (1.7)

4 (2.2)

6 (4.7)

50 (63.3)

Lumbar vertebrae

1 (4.8)

-

1 (4.5)

15 (83.3)

1 (0.8)

-

1 (0.8)

36 (45.6)

Sacro caudal vertebrae

2 (9.5)

1 (3.3)

1 (4.5)

18 (100.0)

2 (1.7)

1 (0.6)

1 (0.8)

67 (84.8)

Thoracic ribs

-

1 (3.3)

2 (9.1)

15 (83.3)

-

1 (0.6)

2 (1.6)

32 (40.5)

Sternum

-

-

3 (13.6)

9 (50.0)

-

-

3 (2.3)

15 (19.0)

Digits

-

-

1 (4.5)

6 (33.3)

-

-

1 (0.8)

14 (17.7)

Long bones (hind limbs)

-

-

1 (4.5)

1 (5.6)

-

-

1 (0.8)

6 (7.6)

Other Changes

Craniofacial malformation

1 (4.8)

-

-

1 (5.6)

1 (0.8)

-

-

1 (1.3)

Domed cranium

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Split cranium

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Reduced lower jaw

1 (4.8)

-

-

-

1 (0.8)

-

-

-

Misshapen clavicles

-

-

-

1 (5.6)

-

-

-

1 (1.3)

Short 13th rib

-

1 (3.3)

1 (4.5)

-

-

1 (0.6)

1 (0.8)

-

Litters and foetuses are counted only once in the totals affected but they may occur in more than one category

Conclusions:
Both maternal and embryo-foetal toxicity was observed at a dose of 1.40 mL/kg bw/day (1428 mg/kg bw/day). Given the physical chemical properties and ability to produce narcosis the embryo-foetal toxic effects were considered to be independent of the maternal toxicity, but likely to stem from the same physical-chemical mechanisms of toxicity. The 0.43 mL/kg bw/day (439 mg/kg bw/day) dose group was considered to be close to the threshold of maternal toxicity, but is considered the NOAEL for maternal toxicity in this study. An enhancement of the incidence of reversible variations and anomalies were recorded in the individual foetuses, litter values however were not affected. No malformations were recorded at this dose level.

Under the conditions of the test, the NOAEL for maternal toxicity, embryo- and developmental toxicity was 0.43 mL/kg bw/day (439 mg/kg bw/day).
Executive summary:

Doses of 1.40, 0.43 and 0.14 mL/kg/day were administered occlusively to the skin to Crl rats between days 6 to 15 of gestation. Maternal animals were observed for signs of toxicity and local irritation. On gestation day 20, the animals were sacrificed and developmental parameters were assessed in the pups in utero. In addition to the developmental toxicity study, an absorption study was included to give a brief picture on the behaviour of the substance when dosed dermally. For this purpose 3 animals from the low and high dose groups received a radiolabelled dose on the final day of treatment.

 

The test material was found to be rapidly and extensively absorbed in the pilot study.

 

Both maternal and embryo-foetal toxicity was observed at a dose of 1.40 mL/kg bw/day (1428 mg/kg bw/day). Given the physical chemical properties and ability to produce narcosis the embryo-foetal toxic effects were considered to be independent of the maternal toxicity, but likely to stem from the same physical-chemical mechanisms of toxicity. The 0.43 mL/kg bw/day (439 mg/kg bw/day) dose group was considered to be close to the threshold of maternal toxicity, but is considered the NOAEL for maternal toxicity in this study. An enhancement of the incidence of reversible variations and anomalies were recorded in the individual foetuses, litter values however were not affected. No malformations were recorded at this dose level.

Under the conditions of the test, the NOAEL for maternal toxicity, embryo- and developmental toxicity was 0.43 mL/kg bw/day (439 mg/kg bw/day).

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
A read across report will be added to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Dose descriptor:
NOAEL
Effect level:
<= 0.43 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: Maternal toxicity
Abnormalities:
no effects observed
Dose descriptor:
NOAEL
Effect level:
> 0.7 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: There was no discernible treatment related effect on developmental toxicity up to the highest dose tested. Concentration equivalent to 710 mg/kg bw/day.
Abnormalities:
no effects observed
Developmental effects observed:
no
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
04 February 1986 to 20 March 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Performed to GLP and reported in a high level of detail, the study is not performed to a standardised guideline and uses lower group numbers than is recommended in OECD 414 guideline, however the study is in basic compliance with the current standardised guidelines and was performed in line with good scientific principles. The study was a range-finding study for a definitive developmental study. Although the study had very specific objectives, all the parameters for assessing developmental toxicity were included. The read-across approach has been used since phenylethyl alcohol is structurally similar to the test material but is more toxicologically active. The results presented are therefore taken to be the worst case scenario.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Undiluted phenethyl alcohol was administered to Crl rats dermally at 0.07, 0.14, 0.28, 0.43 or 0.70 mL/kg/day from gestation day 6 through 15 at 24 hour intervals under an occluded dressing. Emphasis was placed on identifying whether vestigial cervical ribs in offspring were the most sensitive end point for developmental toxicity and whether they occurred in the absence of maternal toxicity. Signs of irritation were noted in all dams dosed with the test material, dose-dependently for onset, severity and duration.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 61 days at receipt. Born on the 05 December 1985 and dosed on 04 March 1986.
- Weight at study initiation: 221-273 g (157 - 205 g on arrival).
- Housing: Individually in wire bottomed stainless steel cages, following cohabitation during mating period.
- Diet: ad libitum.
- Water: Reverse osmosis tap water, ad libitum.
- Acclimation period: 3 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°F): 70 ± 2 ºF
- Humidity (%): 48-58%
- Air changes (per hr): 10 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: From 04 February 1986 to 20 March 1986
Route of administration:
dermal
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: 7 x 5 cm area (shaved) from the intrascapular region.
- Type of wrap if used: Occluded dressing (5 x 5 cm), consisting of an aluminium foil patch (7 x 5 cm) covering all the shaved dorsal area, held in place by a medical-type adhesive bandage.
- Time intervals for shavings or clippings: Shaving was performed when necessary.

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Dosages were applied using computer calculated mL/kg volumes of the neat test material. Up to 0.70 mL/kg bw/day (high dose and control animals).
- Constant volume used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: no
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
The test substance was applied neat, as supplied. The purity of the test substance was confirmed at the beginning and the end of dosing.
Details on mating procedure:
- Impregnation procedure: 130 health females were co-housed with male breeders.
- M/F ratio per cage: 1:1
- Length of cohabitation: Maximum of 4 days.
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of presumed gestation.
Duration of treatment / exposure:
Gestation days 6 to 15.
Frequency of treatment:
Daily
Duration of test:
Up to gestation day 20 (sacrifice).
Remarks:
Doses / Concentrations:
0, 0.07, 0.14, 0.28, 0.43, 0.70 mL/kg/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 71.4, 143, 286, 439 and 714 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
10 female rats were dosed at each test concentration.
Control animals:
other: yes, reverse osmosis water (0.70 mL/kg bw/day)
Details on study design:
- Dose selection rationale: In a previous study, dermal application of the test material to pregnant rats was associated with an increased incidence of fetuses with structural abnormalities. Increased incidences of very small cervical ribs were reported to be the only consistent and reliable indication of altered development at the low (0.14 mL/kg/day) and middle (0.43 mL/kg/day) dosages. The doses selected for this study were chosen to determine whether cervical ribs were the most sensitive indicator of developmental toxicity and whether the cervical ribs occurred in the absence of both maternal toxicity and/or other developmental effects of the test material and whether data appropriate for defining dosages for use in a definitive developmental toxicity study demonstrating both maternal and developmental no-effect levels could be obtained.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily for viability.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Several times during acclimation cohabitation and pre-dosing periods. All mated female rats were evaluated on day 0 of presumed gestation and animals were checked several times daily during administration of the test material (including observations prior to administration and within 30 minutes of application). During the post-dosing period up until the end of the study, animals were examined daily.

BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweights were recorded 4 times prior to the study assignment, on day 0 and daily throughout the dosage and post-dosage periods.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: Feed consumption was recorded for day 0 to 6 of presumed gestation, and daily throughout the dosing and post-dosing periods. Feed utilisation was calculated as g/kg of bodyweight/day.

POST-MORTEM EXAMINATIONS: Yes, animals were assessed for gross external and visceral lesions.
- Sacrifice on gestation day 20.
- Organs examined: Maternal tissues with gross lesions were retained for further assessment, with the exception of the uterus, all other tissues were discarded.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- Foetal weight: Yes: all per litter
- Foetal sex: Yes: all per litter
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third of live foetuses per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: one third of live foetuses per litter
Statistics:
The level of statistical significance for all observations in this study was considered to be P ≤ 0.05.
Maternal clinical signs, pregnancy incidence, abortion, death and total resorption were analysed for homogeneity using the variance test of the binomial distribution.
Maternal bodyweight, bodyweight change, feed consumption and feed utilisation were based on surviving pregnant dams with at least one live pup on day 20. Bartlett’s test of homogeneity of variances and the Analysis of Variance was used to assess maternal bodyweight data and the litter averages for foetal bodyweight, foetal ossification sites, percent dead or resorbed conceptus, percent male foetuses and percent of foetuses with alterations. Where the analysis of Variance was significant, where appropriate the parameters were assessed by Dunnett’s test in order to identity statistical significance of individual groups. If Analysis of Variance was not appropriate, then the Kruskal-Wallis test was used. Where this reached statistical significance, Dunn’s method of multiple comparisons was used to identify the statistical significance of individual groups.
Count data at Caesarean-sectioning were evaluated by the Kruskal-Wallis test. Where these reached statistical significance, Dunn’s method of multiple comparisons was employed to identify statistical significance of individual groups.
The variance test for homogeneity of the binomial distribution was used to analyse all data represented as a proportion.
Indices:
Presence of cervical rib
Average percentage of resorbed conceptuses
Live male foetuses per litter

Foetal alterations were defined as: 1) malformations (irreversible changes); and 2) developmental changes (reversible accelerations or delays of development).
Historical control data:
Historical control data was available for comparison. The historical control data was generated by the testing laboratory between 1983 and 1985.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No maternal animals died during the course of the study.
At 0.70 mL/kg bw/day, ptosis and urine-stained abdominal fur was noted. Ptosis occurred in six (p<0.01) high dosage group dams on a total of 19 days; this sign was not observed in any other test groups in the study. Urine stained abdominal fur was observed for two or three rats in each dosage group. Two high dosage group rats exhibited this for a total of 12 days; this generally occurred no more than twice for the control rats and the rats in the other four dose groups.
No other clinical observation was attributed to administration of the test material. All other clinical signs were single events or were not statistically significant (p>0.05).
Skin reactions were noted at all dose levels with the test material, with increased incidences of rats with low levels (grade 1) of erythema and/or desquamation. The onset, severity and duration were dose-dependent.
At necropsy, one dam in the 0.28 mL/kg/day group and three rats in the 0.70 mL/kg/day group had small lesions present within the application area. In the high dose group the incidence of these skin lesions was significantly increased (p<0.01). The skin lesions were considered effects of dermal application of neat test material.
No other lesions observed at necropsy were attributed to an effect of exposure to the test material, as the incidences were not dosage-dependent or statistically significant.
Administration of the test material did not inhibit average maternal body weight or body weight gain in a consistent dose-dependent manner. Administration of the initial dosage on day 6 of gestation resulted in weight loss (decreased average maternal body weight) in all dose groups, compared with the pre-dosage bodyweights; weight loss was greatest for the control group. The control group had the greatest average weight gain after the second dose was given on day 7 of gestation. A small, inhibitory effect on the average maternal body weight gain occurred for dams given the 0.14 (P<0.01), 0.43 (P > 0.05) and 0.70 mL/kg/day (P<0.01) doses. This response pattern did not persist; no other significant difference (P>0.05) for average maternal body weight change values occurred among the six treatment groups.
Average maternal feed consumption and feed utilization values did not reveal and dose-related patterns. Both were increased after the first and tenth doses were given to dams in all groups administered with the test material compared to the control. For the treated groups, these parameters were decreased, after the third dose was administered. Average maternal feed consumption between days 12-13 of gestation was found to be significantly increased (P<0.05) for the high dose group. There were no other dose-dependent patterns or significant differences.
Pregnancy occurred for in all of the control rats. In the treatment groups, nine, ten, nine, ten and eight of the ten rats per group administered with 0.07, 0.14, 0.28, 0.43 and 0.70 mL/kg bw/day test material, respectively were pregnant. No pregnant dam resorbed all of its litter.
Administration of the test material did not adversely affect the averages for corpora lutea, implantations, litter sizes or resorptions. Values for these parameters were similar and did not reach statistical significance.
Dose descriptor:
NOAEL
Effect level:
<= 0.43 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Abnormalities:
no effects observed
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Litter Data:
Average live foetal bodyweights appeared to be decreased for litters of dams administered with the test substance. This was statistically significant (P < 0.05 to P<0.01) for litters of dams administered with and above 0.14 mL/kg bw/day. However the differences were not biologically significant and were likely a finction of higher numbers of pups/litter in the treated groups.
Administration of the test substance did not affect the average percentage of resorbed conceptuses or of live male foetuses per litter, as compared with control values. Differences observed for these parameters were neither dose—dependent nor significant (P>0.05).
Foetal Alterations:
Foetal alterations were defined as: 1) malformations (irreversible changes); and 2) developmental changes (reversible accelerations or delays of development).

126, 111, 134, 143, 143 and 122 foetuses from 10, 9, 10, 9, 10 and 8 litters from the 0(Vehicle), 0.07, 0.14, 0.28, 0.43 and 0.70 mL/kg bw/day, respectively, were examined for gross external alterations and skeletal alterations and for average numbers of foetal ossification sites. The thoracic and abdominal viscera of 45, 40, 49, 52, 51, and 45 of these respective foetuses were examined for soft tissue alterations.
Administration of 0.43 and 0.70 mL/kg bw/day was found to increase the number of litters with foetal alterations; these increased litter values did not reach statistical significance (P > 0.05). The incidence of altered foetuses was significantly increased (P < 0.05 to P < 0.01) for the two highest dosing groups, as was the average percentage of altered foetuses per litter.
These increased incidences of foetal alteration indicated a dose-dependent pattern of significant increases (P < 0.05 to P < 0.01) in the number of foetuses that had incompletely ossified (hypoplastic) ribs, wavy ribs (0.43 and 0.70 mL/kg bw/day) and/or cervical rib(s) (0.70 ml/kg bw/day only).
Increased incidences of foetuses with delayed ossification of the sternebrae and/or pelvis occurred in all treatment groups. These foetal alterations were not observed for the control group. The litter incidence for foetuses with delayed pelvic ossification was significantly increased (P < 0.05 to P < 0.01) for all dose groups. Neither of these foetal alterations had incidences that demonstrated a clear dose-dependent pattern. Further analysis of the foetal ossification site averages for each litter clearly demonstrated delayed foetal development. Dosage-dependent decreases in ossification site averages occurred for litters of dams in all groups administered with the test substance. The decreases were statistically significant (P < 0.05 to P < 0.01) for metacarpals, hind paw phalanges (0.14 mL/kg bw/day and above), caudal vertebrae (0.28 mL/kg bw/day and above), forepaw phalanges (0.43 mL/kg bw/day and above) and sternal centers (0.07 mL/kg bw/day dosage).
Gross external, soft tissue and/or skeletal examination of the foetuses in this study did not reveal any other foetal alteration attributed the test substance. All other observed foetal alterations (both litter and individual) were not dose-dependent and/or did not reach statistical significance.

Gross External Alterations:
One 0.70 mL/kg bw/day foetus had a thread-like tail.

Soft Tissue Alterations:
No soft tissue alteration was observed for any foetus in this study

Skeletal Alterations:
- Malformation
Cervical rib occurred in 39(32.0%) (P < 0.01) of the foetuses from the eight (100%) (P < 0.01) litters in the high dose group. Differences between the control and those observed for all other dosage groups were not significant (P>0.05). Cervical rib occurred for three (2.4%) foetuses from three (30.0%) control litters and also one 0.07 mL/kg bw/day foetus (foetal incidence = 0.9%; litter incidence = 11.1%). No foetus of the 0.14 mL/kg bw/day dosage group had this alteration. Two (1.4%) foetuses from two (22.2%) litters of the 0.28 mL/kg bw/day group, and five (3.5%) foetuses from two (20.0%) litters of the 0.43 mL/kg bw/day group had cervical rib(s).
No other foetal skeletal malformations were observed.

- Delayed Ossification:
Incompletely ossified (hypoplastic) ribs occurred for 2(1.6%), 1(0.9%), 0, 0, 7(4.9%) (P < 0.05) and 9(7.4%) (P < 0.01) foetuses from 2(20.0%), 1(11.1%), 0, 0, 2(20.0%) and 4(50.0%) litters of the control, 0.07, 0.14, 0.28, 0.43 and 0.70 mL/kg bw/day groups, respectively.
In these same respective groups, wavy ribs occurred for 2(1.6%), 7(6.3%) (P < 0.01), 1(0.7%), 0, 9(6.3%) (P < 0.01) and 11(9.0%) (P < 0.01) foetuses from 2(20.0%), 3(33.3%), 1(10.0%), 0, 4(40.0%) and 4(50.0%) litters.
No control foetuses had incomplete ossification of sternal centers. In the 0.07, 0.15, 0.28 and 0.43 mL/kg bw/day dosage groups, respectively, 7(6.3%) (P < 0.01), 9(6.7%) (P < 0.01), 10(7.0%) (P < 0.01) foetuses and 13(9.1%) (P < 0.01) foetuses from 2(22.2%). 4(40.0%), 4(44.4%) and 5(50.0%) (P < 0.05) litters had one or more incompletely ossified sternal center. One (0.8%) foetus from a 0.70 mL/kg bw/day litter (12.5%) had this alteration.
Delayed ossification of the pelvis (incompletely ossified pubes and/or ischia) was observed only for foetuses of dams administered with the test substance. In the five respective dosage groups, 10(9.0%) (P < 0.01), 7(5.2%), 12(8.4) (P < 0.01), 24(16.8%) (P < 0.01) and 10(8.2%) (P < 0.01) of the foetuses from 3(33.3%) (P < 0.05), 3(30.0%) (P < 0.05), 4(44.4%) (P < 0.01), 8(80.0%) (P < 0.01) and 5(62.5%) (P < 0.01) litters had delayed pelvic ossification.

No other delay in ossification demonstrated a dose-dependent pattern. Low incidences of bifid centra in thoracic vertebra, incomplete ossification of the arches of lumbar vertebrae, incomplete ossification of the manubrium, and unossified sternal centers were recorded but not attributed to the administration of the test substance.

- Foetal Ossification Sites:
The number of ossified metacarpals per foetus averaged 3.74, 3.46, 3.30 (P < 0.01), 3.29 (P < 0.01), 3.26 (P < 0.01) and 3.10(P < 0.01) for the control, 0.07, 0.14, 0.28, 0.43 and 0.70 mL/kg bw/day dose groups, respectively. In these same respective litters, there was an average of 4.56, 3.18, 3.37 (P < 0.05), 3.03 (P < 0.05), 1.67 (P < 0.01) and 1.19 (P < 0.01) ossified hindpaw phalanges per foetus.
In the control, 0.07, 0.14, 0.28, 0.43 and 0.70 mL/kg bw/day dose groups, respectively there was an average of 5.13, 4.66, 4.75, 4.58 (P < 0.05), 4.43 (P < 0.05) and 3.85(P < 0.01) ossified caudal vertebrae per foetus. In these same respective litters, foetuses had an average of 4.92, 3.86, 4.42, 3.97, 2.80 (P < 0.01) and 2.64 (P < 0.01) ossified forepaw phalanges; sternal centers per foetus, the groups averages were 3.80, 3.73, 3.68, 3.70, 3.54 and 3.28 (P < 0.01), respectively.
All other differences in the average numbers of foetal ossification sites per litter were not dose—dependent, were within the expected range for foetuses of this strain and age and/or did not reach statistical significance.

Overall the effects on ossification, along with those on fetal weights is considered to reflect the higher numbers of foetuses per litter in treated groups and greater competition for required nutrients. Overall total litter masses in treated groups tended to be higher than that for the control group.
Dose descriptor:
NOAEL
Effect level:
> 0.7 other: mL/kg bw/day
Based on:
test mat.
Basis for effect level:
other: There was no discernible treatment related effect on developmental toxicity up to the highest dose tested. Concentration equivalent to 710 mg/kg bw/day.
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: Skin Reaction Summary

Dosage (mL/kg/day) Erythema Index Maximum Number Desquamation Index Maximum Number Fissuring Index Maximum Number
0 (vehicle) 16.0 5/10 12.0 3/10 0.0 0/10
7.5 1/10 0.0 0/10 0.0 0/10
0.07 24.0 4/10 20.0 5/10 0.0 0/10
12.5 2/10 22.5 3/10 0.0 0/10
0.14 41.0 7/10 57.0 9/10 3.0 1/10
5.0 2/10 60.0 7/10 0.0 0/10
0.28 43.0 7/10 59.0 7/10 3.0 1/10
50.0 6/10 72.5 6/10 0.0 1/10
0.43 76.0 9/10 80.0 9/10 4.0 2/10
57.5 8/10 102.5 8/10 5.0 1/10
0.70* 120.0 10/10 119.0 10/10 4.0 3/10
130.0 10/10 160.0 10/10 1.0 1/10

* Atonia and eschar were also observed for the high dosage group. During the dosage period, atonia had an index of 1.0 and occurred for 1/10 rats; during the post dosage period, atonia had an index of 10.0 and occurred for 1/10 rats. During the dosage period, eschar had an index of 11.0 and occurred for 3/10 rats; during the postdosage period, eschar had an index of 4.0 and occurred for 2/10 rats. Exfoliation and edema were not observed for rats in any of the dosage groups.

Table 2: Litter Data Summary

  Dosage Group (mL/kg/days 5-15 of presumed gestation)
0 (vehicle) 0.07 0.14 0.28 0.43 0.70
Litters examined day 20 N 10 9 10 9 10 8
Implantations mean± S.D. 13.5± 4.0 12.9 ± 4.9 14.3 ± 4.5 16.7 ± 1.0 15.4 ± 2.1 16.1 ± 1.2
Live foetuses mean± S.D. 12.6 ± 3.8 12.3 ± 4.89 13.4 ± 4.3 15.9 ± 0.8 14.3 ± 2.2 15.2 ± 2.0
Live fetal body weights (g/litter) mean± S.D. 3.40 ± 0.25 3.16 ± 0.22 3.14 ± 0.18* 3.11 ± 0.19* 2.97 ± 0.27** 2.99 ± 0.24**
Male foetuses mean± S.D. 3.49 ± 0.27 3.23 ± 0.27 3.21 ± 0.22* [9] 3.20 ± 0.22* 3.03 ± 0.27** 3.06 ± 0.26**
Female foetuses mean± S.D. 3.30 ± 0.24 3.11 ± 0.20 [8] 3.08 ± 0.19 3.01 ± 0.19* 2.89 ± 0.28** 2.93 ± 0.22**
% resorbed conceptuses/litter mean± S.D. 6.2 ± 6.6  4.1 ± 4.0 5.6 ± 7.0 4.5 ± 4.8 7.2 ± 5.3 5.7 ± 6.4
Live foetuses N 126 111 134 143 143 122
Live male foetuses N 70 54 70 73 82 55
% live male foetuses/total foetuses per litter mean± S.D. 56.2 ± 14.1 53.1 ± 22.4 47.5 ± 18.3 51 ± 8.9 56.8 ± 13.0 45.2 ± 9.8

* Significantly different from the vehicle control at P ≤0.05.

**Significant different from the vehicle control at P ≤ 0.01.

[N] number of values averaged.

Table 3: Fetal Alterations Summary

Dosage Group (mL/kg/days 6-15 of presumed gestation)
0 (vehicle) 0.07 0.14 0.28 0.43 0.70
Litters evaluated N 10 9 10 9 10 8
Foetuses evaluated N 126 111 134 143 143 122
Live foetuses N 126 111 134 143 143 122
Dead foetuses N 0 0 0 0 0 0
Litters with foetuses with any alteration observed N (%) 5 (50.0) 6 (66.7) 5 (50.0) 5 (55.6) 9 (90.0) 8 (100.0)
Foetuses with any alterations observed N (%) 6 (4.8) 20 (18.0) 15 (11.2) 23 (16.1) 44 (30.8)** 59 (48.4)**
% foetuses with an alteration/litter mean ± S.D. 4.40 ± 5.50 14.76 ± 21.54 13.89 ± 18.94 16.21 ± 21.32 30.18 ± 21.60* 49.60 ± 17.42**

* Significantly different from the vehicle control at P ≤0.05.

**Significant different from the vehicle control at P ≤ 0.01.

Table 4: Gross External Fetal-Alterations Summary

  Dosage Group (mL/kg/days 6-15 of presumed gestation)
0 (vehicle) 0.07 0.14 0.28 0.43 0.70
Litters evaluated N 10 9 10 9 10 8
Foetuses evaluated N 126 111 134 143 143 122
Live foetuses N 126 111 134 143 143 122
Dead foetuses N 0 0 0 0 0 0
TAIL:              
Thread-like              
Litter incidence N % 0 0 0 0 0 1 (12.5)
Fetal incidence N % 0 0 0 0 0 1 (0.8)

Table 5: Soft Tissue Fetal Alterations Summary

  Dosage Group (mL/kg/days 6-15 of presumed gestation)
0 (vehicle) 0.07 0.14 0.28 0.43 0.70
Litters evaluated N 10 9 10 9 10 8
Foetuses evaluated N 45 40 48 51 51 45
Live foetuses N 45 40 48 51 51 45
Dead foetuses N 0 0 0 0 0 0

** No soft tissue fetal alterations were found in this study

Table 6: Skeletal Fetal Alterations Summary

  Dosage Group (mL/kg/days 6-15 of presumed gestation)
0 (vehicle) 0.07 0.14 0.28 0.43 0.70
Litters evaluated N 10 9 10 9 10 8
Foetuses evaluated N 126 111 134 143 143 122
Live foetuses N 126 111 134 143 143 122
Dead foetuses N 0 0 0 0 0 0
SKULL:              
Supraoccipital, incompletely ossified              
Litter incidence N % 0 0 1 (10.0) 0 0 0
Fetal Incidence N % 0 0 1 (0.7) 0 0 0
Supraoccipital, not ossified              
Litter incidence N % 0 0 1 (10.0) 0 0 0
Fetal Incidence N % 0 0 1 (0.7) 0 0 0
VERTEBRAE:              
Cervical rib              
Litter incidence N % 3 (30.0) 1 (11.1) 0 2 (22.2) 2 (20.0) 8 (100.0)**
Fetal Incidence N % 3 (2.4) 1 (0.9) 0 2 (1.4) 5 (3.5) 39 (32.0)**
Thoracic, centra, bifid              
Litter incidence N % 1 (10.0) 3 (33.3) 2 (20.0) 0 1 (10.0) 2 (25.0)
Fetal Incidence N % 1 (0.8) 3 (2.7) 2 (1.5) 0 1 (0.7) 2 (1.6)
Lumbar, arch(es), incompletely ossified              
Litter incidence N % 0 0 0 1 (11.1) 0 2 (25.0)
Fetal Incidence N % 0 0 0 1 (0.7)  0 2 (1.6)
RIBS:              
Incompletely ossified (hypoplastic)              
Litter incidence N % 2 (20.0) 1 (11.1) 0 0 2 (20.0) 4 (50.0)
Fetal Incidence N % 2 (1.6) 1 (0.9) 0 0 7 (4.9)* 9 (7.4)**
One or more wavy              
Litter incidence N % 2 (20.0) 3 (33.3) 1 (10.0) 0 4 (40.0) 4 (50.0)
Fetal Incidence N % 2 (1.6) 7 (6.3)** 1 (0.7) 0 9 (6.3)** 11 (9.0)**
MANUBRIUM:              
Incompletely ossified               
Litter incidence N % 0 0 1 (10.0) 1 (11.1) 1 (10.0) 0
Fetal Incidence N % 0 0 1 (0.7) 1 (0.7) 1 (0.7) 0
STERNABRAE:              
One or more incompletely ossified              
Litter incidence N % 0 2 (22.2) 4 (40.0) 4 (44.4) 5 (50.0) 1 (12.5)
Fetal Incidence N % 0 7 (6.3)** 9 (6.7)** 10 (7.0)** 13 (9.1)** 1 (0.8)
One or more not ossified              
Litter incidence N % 0 1 (11.1) 2 (20.0) 1 (11.1) 2 (20.0) 3 (37.5)
Fetal Incidence N % 0 2 (1.8) 3 (2.2) 2 (1.4) 4 (2.8) 3 (2.4)
PELVIS:              
Pubes and/or ischia, incompletely ossified              
Litter incidence N % 0 3 (33.3)* 3 (30.0)* 4 (44.4)** 8 (80.0)** 5 (62.5)**
Fetal Incidence N % 0 10 (9.0)** 7 (5.2) 12 (8.4)** 24 (16.8)** 10 (8.2)**
Pubes and/or ischia, not ossified              
Litter incidence N % 0 0 1 (10.0) 0 2 (20.0) 1 (12.5)
Fetal Incidence N % 0 0 1 (0.7) 0 2 (1.4) 1 (0.8)

* Significantly different from the vehicle control at P ≤0.05.

**Significant different from the vehicle control at P ≤ 0.01.

Conclusions:
Dosage dependent increasing scores for dermal irritation resulted from administration of all dosages of the test material as compared with control values. In addition, the 0.7 mL/kg/day dosage of the test material caused incidences of ptosis and/or urine-stained abdominal fur in the dams. On the basis of these findings, the neat test material was considered irritating to the skin of pregnant rats. Secondary effects of this irritation may have resulted in the observed altered fetal development.

Dosage dependent statistically significant observations of delays in fetal development (decreased average fetal body weight and delayed fetal ossification) resulted from administration of the test material at 0.14 mL/kg/day dosages and above. There were no treatment related soft tissue effects in foetuses. Significant increase in the incidence of foetuses with cervical ribs occurred for the 0.70 mL/kg/day group. All other foetal observations were considered to be reversible delays in ossification. Delayed ossification (sternum and/or pelvic) was noted in the all test material administered groups. Delayed pelvic ossification was found to be significant in all groups dosed with the test material, but neither alteration demonstrated a clear dosage dependent pattern. No skeletal malformations were observed.

Under the conditions of the study, on the basis of no adverse fetal effects, and ptosis and/or urine-stained abdominal fur in dams developmental and maternal no-effect levels for the test material were 0.7 and ≤ 0.43 mL/kg/day respectively, equivalent to 710 and ≤ 439 mg/kg bw/day.
Executive summary:
The developmental toxicity of phenylethyl alcohol was investigated by administering undiluted phenethyl alcohol to rats dermally at 0.07, 0.14, 0.28, 0.43 or 0.70 mL/kg/day from gestation day 6 through 15 at 24 hour intervals under an occluded dressing. Signs of irritation were noted in all dams dosed with the test material, dose-dependently for onset, severity and duration. No maternal deaths occurred. Clinical signs of toxicity (ptosis and/or urine stained abdominal fur) were noted in the dams dosed with 0.70 mL.kg bw/day of the test material. Live foetal body weights were statistically significantly lower in litters from dams dosed with the test material, but these were not biologically significant and likely a function of the increased numbers of fetuses per litter in treated groups. Significant increase in the incidence of foetuses with cervical ribs occurred for the 0.70 mL/kg/day group. These are reversible as are perturbations/delays in ossification caused by increased competition for nutrients as a result of increased foetal numbers in treated groups. Often the delays or perturbations in ossification were not treatment related. Under the conditions of the study, on the basis of no discernible adverse effects on fetal development, and ptosis and/or urine-stained abdominal fur in dams (and excluding irritation), the developmental and maternal no-effect levels for the test material were 0.7 and ≤0.43 mL/kg/day respectively.
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August 2021 to 21 December 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: “Regulation on Test Methods for Chemical Substances” Notification No. 2020-46 National Institute of Environmental Research, Republic of Korea
Version / remarks:
03 November 2020
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD), SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males were 10 weeks old. Females were 9 weeks old at receipt and 10 - 11 weeks old at start of administration.
- Weight at study initiation: Males were 305.5 – 381.1 g. Females were 195.8 – 234.0 g at receipt and 250 - 311.7 g at start of administration.
- Fasting period before study: No
- Housing: Animals were housed in stainless wire mesh cages, 260 W × 350 D × 210 H (mm) (excluding use of polycarbonate cages). Then from GD 16 to necropsy animals were housed in polycarbonate cages, 260 W × 420 D × 180 H (mm). One – three animals were housed per cage during the quarantine acclimation period and females were housed individually during the gestation period.
- Diet: Powder feed rodent chow was placed in feeders and provided ad libitum.
- Water: Public tap water was filtered and irradiated by a UV water steriliser and then provided ad libitum.
- Acclimation period: 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4 – 22.8 °C.
- Humidity (%): 55.4 – 73.2 % (relative)
- Air changes (per hr): 10 – 15 clean, fresh, filtered air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/ dark cycle (7 AM to 7 PM via automated timer), 150 - 300 Lux.

IN-LIFE DATES:
From: 10 August 2021
To: 12 September 2021
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test material was weighed and placed in a container. The required amount of vehicle was added and mixed using a vortex mixer until dissolved.
The required amount of powder feed except for the amount of the test material was weighed. The required amount of the test material formulation and a small amount of powder feed were mixed in a container. The mixture was then placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration.
The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5 – 10 minutes for the control group.

DIET PREPARATION
- Storage temperature of food: The dosing feed was stored under refrigeration conditions for use within 14 days and used at room temperature (1 – 30 °C) for 6 days.

VEHICLE
- Concentration in vehicle: 10 mL of vehicle for 1 kg of powder feed containing test material.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis for homogeneity and stability of the dosing formulations was conducted. The homogeneity and stability at room temperature (1 – 30 °C) for 3 and 6 days and under refrigeration for 8 and 14 days of dosing formulations were confirmed for the dose levels of 0.04 and 1.5 %.
Samples were taken three times from the middle of each dosing formulation and analysed for verification of dose level concentration prior to the first dosing. As a result, the accuracies at 0.25, 0.5 and 1.0 % were 97.92, 95.60 and 96.30 % prior to dosing, respectively. These results were within the acceptable range (range: ± 15 % of nominal values).

DETAILS ON ANALYTICAL METHODS
- Standard Substance
The test substance was used as the standard substance for the comparison of analytical results.

- Vehicle
Corn oil

- Analytical Instrumentation and Equipment
Gas Chromatography (GC):
System: GC-2010 series, Shimadzu Corp., Japan
Main body: GC-2010AF
Autosampler: AOC-20s
Autoinjector: AOC-20i
Detector: Flame Ionization Detector (FID)
Data processor: GC solution ver. 2.3

- Solution for Dilution
tert-Butyl methyl ether was used as the solution for dilution.

- Preparation of the Standard Solutions
Standard stock solution of the highest concentration was prepared by weighing 0.1 g of the standard substance in a 100 mL volumetric flask and diluting with the solution for dilution (concentration: 0.1 %).
Other standard solutions were prepared by diluting this standard stock solution of high concentration with the solution for dilution yielding the concentrations of 0.001, 0.002, 0.005, 0.01 and 0.02 %.

- Quality Control (QC) Sample
The standard solution at the concentration of 0.005 % was used as a QC sample.

- Preparation of Dosing Formulations
The required amount (14.996 g) of the test material was weighed and placed in a container. The required amount of vehicle (10 mL of vehicle for 1 kg of powder feed containing test material) was added and mixed using a vortex mixer until dissolved. The required amount (1 000.04 and 985.48 g) of powder feed except for the amount of the test material was weighed. The required amount of the test material formulation and a small amount of powder feed were mixed in a bottle. The mixture was then placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration. The dosing formulations at concentrations of 0.04 and 1.5 % were prepared for analyses for homogeneity and stability at room temperature (1 – 30 °C) for 3, 6 and 8 days and under refrigeration (at 2 − 8 °C) for 8 and 14 days.

- GC Analytical Conditions
Column: ZB-1ms column (20 m × 0.18 mm, 0.18 μm, Phenomenex, U.S.A); Flow: 1.61 mL/min; Oven: A temperature of 60 °C with a hold time of 3 minutes and a temperature of 240 °C with a hold time of 3 minutes at a rate of 9 °C per minute.
Injector: Temperature 200 °C; Split: 70:1; Injection volume: 1 μL
Carrier gas: Nitrogen
Gas flow rate: Makeup gas (Nitrogen): 30 mL/min; Hydrogen gas: 40 mL/min; Air: 400 mL/min
Run time: 26 min
Detector Temperature: 275 °C

- Treatment of the Dosing Formulations
The required amount (1 g) of the samples was taken from each part of the dosing formulations. The 0.04 and 1.5 % samples were diluted with the solution for dilution by applying dilution factors of 10 and 100, respectively, and filtered through a 0.45 μm PTFE-H syringe. Then, 1 μL was injected into the GC within the concentration range of calibration samples and analysed for homogeneity and stability at room temperature (1 – 30 °C) for 3, 6 and 8 days and under refrigeration (at 2 − 8 °C) for 8 and 14 days.

- Calculations
Linear regression
The calibration curve was constructed by plotting the peak area of the standard solution versus the concentration of standard solution. The linear regression equation was:

y = ax + b

Where:
y = Peak area generated by each standard solution
x = Concentration of the analyte in each standard solution
a = Slope of the calibration curve
b = Y-intercept of the calibration curve

Sample concentration
The measured concentration of the analyte in the samples was determined using the calibration curve (y = ax + b) and by solving the x variable:

x = [(y - b) / a] x d

Where:
y = Peak area generated by the sample
x = Measured concentration of the analyte in each sample
a = Slope of the calibration curve
b = Y-intercept of the calibration curve
d = Dilution factor

Precision, accuracy and variation were calculated as follows:
Precision (%) = (Standard deviation of determined concentrations / Mean of determined concentrations) × 100
Accuracy (%) = (Mean of determined concentrations / Nominal concentration) × 100
Variation (%) = [(Mean value after storage – Mean value of initial concentrations) / Mean value of initial concentrations] × 100

ANALYSES OF THE DOSING FORMULATIONS
- System Suitability
The QC sample was analysed 6 times to evaluate the precision. The values were considered to be acceptable when the precision of peak area and retention time were 3 % or less.

-Linearity
The standard solutions of concentrations from 0.001 to 0.02 % were analysed. The correlation coefficient between the concentration and the peak area of the standard solutions was calculated. The calibration curve was used to evaluate the results of stability analysis of the dosing formulations.
The results were judged to be acceptable when the correlation coefficient (r) of the calibration curve was more than 0.9950 and the accuracy of each concentration was in the range of 85 − 115 % of theoretical values.

- Specificity
The solutions for dilution and vehicle were analysed to check for interfering peaks at the retention time of the standard substance to confirm assay specificity.
The results were judged to be acceptable when a sufficient number of peaks of the standard substance was observed and there were no interfering peaks at the same retention time as the standard substance.

- Intra-day Variation
The samples from the initial point of the dosing formulations of each batch were collected in triplicate and analysed once each.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 80 – 120 % of theoretical values.

- Stability of the Stock Solution
The stock solution was stored under refrigeration for 3 and 8 days. Samples were prepared in triplicate at the concentration of QC sample and analysed.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 85 − 115 % of theoretical values.

- Stability in Autosampler
The samples analysed for intra-day variation were allowed to stand for a certain period of time in the autosampler and re-analysis conducted to check for stability in the autosampler.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.

- Homogeneity
The dosing formulations were randomly divided into 3 points, collected in triplicate from each point and analysed once per sample. The result of the initial point was used as a result of intra-day variation.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 80 − 120 % of theoretical values.

- Stability
Stability following 3 ,6 and 8 days of standing at room temperature (1 – 30 °C): All dosing formulations were allowed to stand for 3, 6 and 8 days at room temperature and three replicate samples from the initial point of dosing formulations of each batch were analysed for stability. The stability of the low dose formulation at room temperature for 8 days was outside the acceptance criteria, thus the analysis for stability at room temperature for 14 days was not performed.
The results of intra-day variation were used as the results of analysis performed immediately after preparation.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.
Stability following 8 and 14 days storage under refrigeration: All dosing formulations were stored under refrigeration for 8 and 14 days and three replicate samples from the initial point of dosing formulations of each batch were analysed for stability.
The results of intra-day variation were used as the results of analysis performed immediately after preparation.
The results were judged to be acceptable when the precision was 10 % or less and the variation of stability was within ± 15 % of the initial concentration.

- Quality Control (QC) Sample
The QC sample was analysed 3 times after the completion of other analysis to confirm the condition of equipment and analytical method.
The results were judged to be acceptable when the precision was 10 % or less and the accuracy was in the range of 85 − 115 % of theoretical values.

RESULTS
- System Suitability: The precision results of peak area and retention time of 0.005 % QC sample analysed 6 times were 1.05 and 0.08 %, respectively.
- Linearity: The correlation coefficient of the calibration curves in the concentration range of 0.001 to 0.02 % of the standard solution was 1.0000 and the accuracy of each concentration was 98.98 – 104.10 % on Day 0, 97.40 – 101.10 % on Day 3 and 98.90 – 101.00 % on Day 6, 99.00 – 102.35 % on Day 8 and 98.48 – 106.10 % on Day 14.
- Specificity: The chromatogram of the low standard solution showed enough peaks which were possible to analyse, and chromatograms of the solutions for dilution and vehicle did not reveal any interfering peaks at the same retention time for the standard substance.
- Intra-day Variation: The precision results of intra-day variation were 0.81 and 0.21 % for 0.04 and 1.5 % of dosing formulations, respectively. The accuracy results were 98.30 and 93.87 %, respectively.
- Stability of the Stock Solution: Stock solution was stored under refrigeration for 3 and 8 days. The precision results were 0.04 and 0.16 % at 0.005 % of the standard solution, respectively. The accuracy results were 99.90 and 100.82 %, respectively.
- Stability in the Autosampler: The precision results of reanalysed intra-day variation samples in the autosampler for 9 hours were 1.29 and 0.28 % for 0.04 and 1.5 % of dosing formulations, respectively. The variation results were 0.66 and 0.28 %, respectively.
- Homogeneity: The precision results of homogeneity were 0.89 and 0.35 % for 0.04 and 1.5 % of dosing formulations, respectively. The accuracy results were 98.48 and 94.07 %, respectively.
- Stability
Stability following 3 days of standing at room temperature (1 – 30 ℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.48 and 0.51 %, respectively, for 3 days standing at room temperature. The variation results were -5.06 and -3.05 %, respectively.
Stability following 6 days of standing at room temperature (1 – 30 ℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.29 and 0.39 %, respectively, for 6 days standing at room temperature. The variation results were -11.88 and -8.31 %, respectively.
Stability following 8 days of standing at room temperature (1 – 30℃): The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.97 and 1.26 %, respectively, for 8 days standing at room temperature. The variation results were -16.48 and -9.52 %, respectively.
Stability following 8 days of storage under refrigeration: The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.99 and 0.64 %, respectively, for 8 days stored under refrigeration. The variation results were -2.14 and 0.57 %, respectively.
Stability following 14 days of storage under refrigeration: The precision results of dosing formulations for 0.04 and 1.5 % to verify stability were 0.22 and 0.43 %, respectively, for 14 days stored under refrigeration. The variation results were 3.61 and -1.99 %, respectively.
- Quality Control (QC) sample: At the completion of analysis, three replicate 0.005 % triplicate QC samples were analysed. The precision and accuracy results were 1.24 and 98.72 % on Day 0, 0.45 and 98.36 % on Day 3, 0.88 and 99.46 % on Day 6, 0.76 and 97.84 % on Day 8 and 0.60 and 99.66 % on Day 14.

DISCUSSION AND CONCLUSION
The gas chromatography (GC) method for the analysis of the dosing formulations was validated. The stability of the low dose formulation at room temperature for 8 days was outside the acceptance criteria, thus the analysis for stability at room temperature for 14 days were not performed. In conclusion, the 0.04 and 1.5 % dosing formulation was homogeneous in corn oil and stable after standing at room temperature (1 – 30 °C) for 3 and 6 days and under refrigeration for 8 and 14 days.
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
- M/F ratio per cage: 1:1. During the mating period, virgin female rats were cohabitated with male breeder rats (one male rat per one female rat) in the home cages housing sexually mature breeder males. Female rats without evidence of mating were separated from the male rats in the morning and individually housed until that afternoon. Then, the male and female rats were cohabitated again in the afternoon.
- Length of cohabitation: The mating period was performed for seven days. Then, mating was ended when 20 female rats per group are copulated.
- Proof of pregnancy: Confirmation of pregnancy was conducted the next morning. Mating was evaluated daily during the cohabitation period. The day when female rats were observed with spermatozoa in a smear of the vaginal contents and/or the presence of a copulatory plug was observed in situ was considered to be GD 0.
Duration of treatment / exposure:
Gestation Day (GD) 5 to GD 19.
Frequency of treatment:
Daily. Mixed test material formulation and powder feed were placed in feeders and provided ad libitum.
Duration of test:
GD 5 to GD 19, total 15 days.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 %
Dose / conc.:
221.3 mg/kg bw/day (nominal)
Remarks:
0.25 %
Dose / conc.:
454.6 mg/kg bw/day (nominal)
Remarks:
0.5 %
Dose / conc.:
925.1 mg/kg bw/day (nominal)
Remarks:
1.0 %
No. of animals per sex per dose:
20 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a result of the dose range finding study, no treatment-related changes were observed in any doses tested. In addition, no deaths were observed at a dose of 1.5 %. And, mean intake test material in the high dose group (1.5 %) was observed at 1 446.7 mg/kg/day. The expected mean intake test material in the high dose group is approximately 1 000 mg/kg/day, 1.0 % was selected as the high dose of this study. The mid and low doses were selected at 0.5 and 0.25 %, respectively. Control animals were received the standard basal powder feed with corn oil (vehicle).
- Rationale for animal assignment: On the day following mating, copulated females with body weights close to the mean body weight were selected and assigned to 4 groups (20 animals/ group). Selected animals were randomly assigned to achieve an even distribution of mean body weight of each group.
- Fasting period before blood sampling for (rat) dam thyroid hormones: No fasting.
- Time of day for (rat) dam blood sampling: Blood sampling took place after necropsy.
Maternal examinations:
The parameters listed below were evaluated for all copulated females. However, pregnancy was only finally confirmed at the time of caesarean section; non-pregnant animals were not reflected in the final report.

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs and twice daily for mortality, moribundity, abortion and premature birth during the observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GDs 0, 3, 5, 8, 11, 14, 17, 19 and 20 (the day of necropsy) using an automatic animal balance.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: For the determination of food consumption for individual animals, feed of measured quantity was offered on GDs 0, 2, 4, 5, 7, 10, 13, 16 and 18, and the amount of feed left was measured on the respective following day.
- Food consumption: Individual food consumption data was determined by subtracting the amount of feed left from the given quantity.
- Test substance intake: The test substance intake was calculated as follows and designated by mg/kg/day:
Test substance intake (mg/kg/day) = [Food consumption (mg/day) × Concentration (%)] / Body weight (kg)

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20. All animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anaesthesia on the day of necropsy.
- Gross pathology examinations: Complete gross post-mortem examinations were performed on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.
- Organ weights: The following organs of all pregnant animals were harvested and wet weight was measured, and the relative ratio of organ weight to terminal body weight of dams (excluding gravid uterus with cervix weight) was calculated: Gravid uterus with cervix, thyroid gland with parathyroid gland (weighed paired and after fixation) and liver. Paired organs were weighed together.
- Histopathology: At necropsy, the following organs and tissues from all pregnant animals were harvested and preserved in 10 % neutral buffered formalin: Brain, pituitary gland, ovary, uterus with cervix, liver, parathyroid gland and thyroid gland.
Histopathological examination of the samples of the following organs and tissues from all pregnant animals was performed: Liver, thyroid gland and parathyroid gland. The parathyroid specimens were examined histopathologically only if present in routine sections.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

At necropsy, the ovaries and uterus of all females were removed and implantations, early and late resorptions and dead/live foetuses were examined; the number of corpora lutea and implantation sites were counted. The pre- & post-implantation loss rates, early & late resorptions rates and embryofoetal mortality rate were calculated.
Blood sampling:
- Thyroid hormone analysis: Blood samples from the abdominal aorta of animals were taken within a short time (two hours) on the morning of the day of necropsy. Collected blood samples were centrifuged at 3 000 rpm for 10 minutes to obtain serum. The Triiodothyronine (T3), Total thyroxine (T4) and Thyroid stimulating hormone (TSH) from separated serum were analysed using an immunoassay analyser.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
- Head examinations: No
- Anogenital distance of all pups: Yes

- Sex ratio: The sex of each foetus was determined by comparing the distance between the anus and the genital tubercle (short for females, long for males). The sex ratio was calculated as follows:

- External examination of foetuses and observation of placenta: All foetuses were examined for external findings including the eyes, ears, nose, mouth, palate, absence of limbs and tail, position and size, shape etc. In addition, the shape and colour of the placenta were observed.

- Body weights of live foetuses and placenta weights: Live foetuses removed from the uterus were numbered sequentially from the left ovary to the right ovary on the dorsal surface with an indelible pen. The weights of surviving foetuses and placental weights were recorded. Foetal body weights and placental weights were based on the average value of the foetuses of the same litter. All live foetuses were euthanised by hypothermia.

- Anogenital distance: AGD of each foetus was measured after caesarean section using a Digimatic calliper.

- Visceral examination of live foetuses: For the visceral examination of foetuses, specimens of the preserved odd number foetuses were prepared by FA solution. Then, microdissections were examined for the visceral examination using a stereomicroscope. All specimens and sections were individually preserved in 10 % neutral buffered formalin.

- Skeletal examination of live foetuses: For the skeletal examination of foetuses, specimens of the preserved even number foetuses were prepared. The skeletons were stained using Alizarin Red S by the single staining technique. Subsequently, these foetuses were evaluated using a stereomicroscope and/or macroscopic examination for skeletal anomalies and ossification variations. These specimens were then stored in 98 - 100 % glycerin.
Statistics:
Statistical analysis was performed for the body weights of live pregnant animals, food consumption, thyroid hormone values, organ weights, number of corpora lutea, number of implantations, pre- & post- implantation rates, body weights and placenta weights of live foetuses, number of live foetuses, sex ratio, number of ossifications, and AGD index using the SAS program (version 9.4, SAS Institute Inc., U.S.A.).
The data was analysed utilizing Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data (significance level: 0.05); then, if significant, Dunnett's test was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed). The Kruskal-Wallis test was employed on heterogeneous data (significance level: 0.05); then, if significant, DSCF (Dwass-Steel-Critchlow-Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
The results of other data types such as malformation and anomalies through skeletal examination, visceral examination and external findings, early resorption rate, late resorption rate and embryofoetal mortality rate were analysed utilising the Kruskal-Wallis test (significance level: 0.05). If significant, DSCF (Dwass-Steel-Critchlow-Fligner) was applied for multiple comparisons (significance levels: 0.05 and 0.01, two-tailed).
Indices:
- Pre-implantation loss rate (%) = [(No. of corpora lutea – No. of implantations) / No. of corpora lutea] × 100
- Post-implantation loss rate (%) = [(No. of implantations – No. of live foetuses ) / No. of implantations] × 100
- Early resorptions (%) = (No. of early resorptions / No. of implantations) × 100
- Late resorptions (%) = (No. of late resorptions / No. of implantations) × 100
- Foetal mortality (%) = ((No. of resorptions + No. of dead foetuses) / No. of implantations) × 100
- Anogenital Distance index: AGD index was calculated as follows: AGD index = AGD / cubed root of body weight
- Sex ratio (%) = (No. of live male foetuses / No. of total live foetuses) × 100
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Crushing of teeth was observed in one animal and overgrown teeth were observed in another in the 0.25 % group. It was not considered to be a test material-related effect since these were low incidence, not dose-related, or comparable to the vehicle control animals, and considered incidental or spontaneous.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths in the animals in the control group, 0.25, 0.5 and 1.0 % groups during the observation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test material-related effects in body weights and body weight gains were observed in the 0.25, 0.5 and 1.0 % groups during the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test material-related effects in food consumption and relative food consumption were observed in the 0.25, 0.5 and 1.0 % groups during the study period.
The mean total test material intakes in the 0.25, 0.5 and 1.0 % groups were 221.3, 454.6 and 925.1 mg/kg/day, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
no effects observed
Description (incidence and severity):
No test material-related effects in thyroid hormone analysis were observed in the 0.25, 0.5 and 1.0 % groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No test material-related effects in absolute and relative organ weights were observed in the 0.25, 0.5 and 1.0 % groups.
No test material-related effect in placental weights was observed in the 0.25, 0.5 and 1.0 % groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no noticeable findings were observed in the control, 0.25, 0.5 and 1.0 % groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination did not reveal treatment-related changes in all pregnant females in the 0.25, 0.5 and 1.0 % groups. All microscopic findings seen in the liver, thyroid gland and parathyroid gland did not show significant difference in the incidence compared to controls or were normal background lesions observed at the testing facility or were found with low/ isolated frequency.
Histopathological findings: neoplastic:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test material-related changes in caesarean section parameters were observed in the 0.25, 0.5 and 1.0 % groups.
No test material-related effect in placentas was observed in the 0.25, 0.5 and 1.0 % groups. The changes including shared, large and small placentas in the control, 0.25 and 0.5 % groups were observed without dose-dependence. Therefore, these changes were not considered to be test material related.
No test material-related effect in placental weights was observed in the 0.25, 0.5 and 1.0 % groups.
Key result
Dose descriptor:
NOAEL
Effect level:
925.1 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: No adverse effects were observed at the 1.0 % dose level, the highest tested.
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test material-related effect in foetal body weights was observed in the 0.25, 0.5 and 1.0 % groups.
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
not specified
Anogenital distance of all rodent fetuses:
no effects observed
Description (incidence and severity):
No test material-related effect in AGD index of male and female foetuses was observed in the 0.25, 0.5 and 1.0 % groups.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
No test material-related effect in external examinations of foetuses was observed in the 0.25, 0.5 and 1.0 % groups.
Skeletal malformations:
no effects observed
Description (incidence and severity):
No test material-related effects were observed in the skeletal examination of foetuses in the study.
No skeletal malformation was observed in any groups.
The skeletal variations observed in the study were not considered to be test material-related effects because these were not statistically significant and/ or were not dose dependent.
No test material-related effects in the number of ossification centres were observed in this study.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test material-related effects were observed in the visceral examination of foetuses in the study.
No visceral malformation was observed in any groups.
The visceral variations observed in the study were not considered to be test material-related effects because these were not statistically significant and/ or were not dose dependent.
Key result
Dose descriptor:
NOAEL
Effect level:
925.1 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed at the 1.0 % dose level, the highest tested.
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no

Summary of Clinical Signs: Females

Group/ Dose

(%)

No. of Animals

Clinical Sign

Gestation Day

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

G1: 0

19

NOA

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

19

NOA

19

19

19

19

19

19

19

19

19

19

19

19

19

19

17

19

19

19

19

19

Crushing of teeth

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

 

 

Overgrown teeth

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1

 

 

 

 

 

G3: 0.5

17

NOA

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

20

NOA

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

20

NOA: No Observable Anomaly.

 

Mean Body Weights: Females (g)

Group/ Dose

(%)

Gestation Day

0

3

5

8

11

14

17

19

20

G1: 0

Mean

249.9

271.8

282.1

294.5

314.0

332.2

363.2

395.2

412.8

S.D

9.9

12.0

12.8

14.8

15.4

16.9

18.7

23.0

23.5

N

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

249.6

270.8

279.4

292.7

311.3

331.1

356.9

389.1

405.9

S.D

11.9

11.8

12.3

13.4

15.3

15.0

22.6

26.2

32.2

N

19

19

19

19

19

19

19

19

19

G3: 0.5

Mean

250.9

271.7

281.1

295.8

315.5

332.4

362.4

395.8

416.0

S.D

12.5

12.1

11.8

12.1

14.3

12.7

17.6

21.4

22.9

N

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

249.3

272.3

282.8

297.5

316.1

333.1

365.8

398.2

416.6

S.D

12.0

132

13.7

15.5

18.2

18.0

18.9

21.6

23.7

N

20

20

20

20

20

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Body Weight Gains: Female (g)

Group/ Dose

(%)

Gestation Day

3

5

8

11

14

17

19

20

G1: 0

Mean

21.8

10.4

12.4

19.4

18.2

31.1

32.0

17.6

S.D

5.6

3.6

5.0

5.0

4.6

6.1

6.2

5.1

N

19

19

19

19

19

19

19

19

G2: 0.25

Mean

21.1

8.7

13.3

18.6

19.9

25.8

32.1

16.8

S.D

6.7

3.9

4.8

5.7

5.5

14.1

12.8

7.7

N

19

19

19

19

19

19

19

19

G3: 0.5

Mean

20.8

9.5

14.7

19.6

16.9

30.1

33.4

17.2

S.D

4.3

2.8

3.5

4.9

5.5

7.9

6.5

7.2

N

17

17

17

17

17

17

17

17

G4: 1.0

Mean

23.0

105

14.7

18.6

17

32.7

32.4

18.4

S.D

4.8

3.8

4.7

6.2

6.4

7.7

5.3

10.9

N

20

20

20

20

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Food Consumption: Female (g/day)

Group/ Dose

(%)

Gestation Day

1

3

5

6

8

11

14

17

19

G1: 0

Mean

23.4

25.1

26.3

27.2

27.4^

28.4

27.8^

33.7^

32.4^

S.D

3.1

3.3

3.0

3.3

3.8

3.1

2.6

5.4

3.7

N

19

19

19

19

18

19

18

16

16

G2: 0.25

Mean

21.8

25.2

25.1^

26.3^

27.1^

28.5

28.2^

30.4^

30.9^

S.D

2.8

3.2

2.9

3.5

3.6

3.4

3.7

4.9

4.3

N

19

19

18

18

18

19

18

17

17

G3: 0.5

Mean

32.2

25.5^

26.5^

27.1^

27.5

29.4

29.4^

32.6^

33.2

S.D

4.6

4.3

3.7

4.5

3.4

3.5

2.7

5.1

3.9

N

17

16

16

16

17

17

16

16

17

G4: 1.0

Mean

23.1^

26.6^

27.8^

29.2^

28.7^

29.1

29.5^

32.8^

33.8^

S.D

4.3

4.6

4.2

5.1

4.3

3.7

2.9

4.8

4.1

N

19

19

19

16

18

20

17

15

19

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

 

Mean Relative Food Consumption: Female (g/kg body weight/day)

Group/ Dose

(%)

Gestation Day

1

3

5

8

11

14

17

19

G1: 0

Mean

93.7

92.1

93.0

92.6^

90.4

83.6^

93.5^

83.1^

S.D

12.9

10.9

9.0

11.0

7.5

6.3

16.1

11.5

N

19

19

19

18

19

18

16

16

G2: 0.25

Mean

97.5

93.2

89.8^^

92.3

91.6

85.1^

85.5^

80.3^

S.D

12.6

11.8

9.8

10.8

9.3

10.6

13.0

11.2

N

19

19

18

18

19

18

17

17

G3: 0.5

Mean

92.8

94.1^

94.6^

92.9

93.3

88.8^

90.1^

83.8

S.D

18.5

16.5

13.7

11.1

9.9

8.5

14.0

8.1

N

17

16

16

17

17

16

16

17

G4: 1.0

Mean

92.5^

97.7^

98.1^

96.5^

91.9

88.4^

89.2^

85.0^

S.D

15.5

14.9

122

12.9

9.3

7.6

11.7

9.5

N

19

19

19

18

20

17

15

19

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

Mean Test Material Intake: Female (mg/kg/day)

Group/ Dose

(%)

Gestation Day

Total Mean*

6

8

11

14

17

19

G2: 0.25

Mean

235.0^

230.7^

229.1

212.8^

214.0^

200.7^

221.3

S.D

28.5

27.1

23.2

26.6

32.5

28.1

17.3

N

18

18

19

18

17

17

19

G3: 0.5

Mean

483.4^

464.4

466.5

443.6

450.8

418.9

454.6

S.D

79.7

55.3

49.7

42.8

70.1

40.5

38.3

N

16

17

17

16

16

17

17

G4: 1.0

Mean

1 040.7^

965.4^

918.6

884.0^

891.8^

849.5^

925.1

S.D

158.5

129.1

92.6

76.2

117.3

211.4

64.716

N

16

18

20

17

15

19

20

^: The animals that abandoned most of the feed from the feeder was excluded from the data.

*: The animals that abandoned most of the feed from the feeder was excluded from the total mean data.

 

Mean Thyroid Hormone Value: Female

Group/ Dose

(%)

T4

(μg/dL)

TSH

(μIU/mL)

T3

(ng/dL)

G1: 0

Mean

0.93

0.248

37

S.D

0.41

0.112

9

N

19

19

19

G2: 0.25

Mean

1.14

0.273

40

S.D

0.83

0.243

16

N

19

19

19

G3: 0.5

Mean

1.04

0.186

42

S.D

0.36

0.181

11

N

17

17

17

G4: 1.0

Mean

0.99

0.225

43

S.D

0.60

0.083

21

N

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Absolute Organ Weights: Female (g)

Group/ Dose

(%)

B.W

Liver

Gravid Uterus with Cervix

Thyroid Gland with Parathyroid Gland

G1: 0

Mean

412.8

16.00

82.88

0.0293

S.D

23.5

1.22

13.93

0.003

N

19

19

19

19

G2: 0.25

Mean

405.9

15.77

77.83

0.315

S.D

32.2

2.11

22.32

0.0050

N

19

19

19

19

G3: 0.5

Mean

413.0

16.05

81.85

0.0288

S.D

22.9

1.24

11.27

0.0055

N

17

17

17

17

G4: 1.0

Mean

416.6

16.39

84.15

0.0311

S.D

23.7

1.66

9.44

0.0056

N

20

20

20

20

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Relative Organ Weights: Females (g/ 100g body weight)

Group/ Dose

(%)

Correct B.W

Liver

Gravid Uterus with Cervix

Thyroid Gland with Parathyroid Gland

G1: 0

Mean

329.9

3.8757

25.1051

0.0071

S.D

14.6

0.1820

3.9610

0.0012

N

19

19

19

19

G2: 0.25

Mean

328.1

3.8708

23.7007

0.0078

S.D

18.8

0.2782

6.9631

0.0012

N

19

19

19

19

G3: 0.5

Mean

331.1

3.8865

24.7298

0.0070

S.D

16.8

0.2040

3.2280

0.0014

N

17

17

17

17

G4: 1.0

Mean

332

3.9279

25.3167

0.0075

S.D

17.9

0.2510

2.4745

0.0014

N

20

20

20

20

Correct B.W. = Terminal B.W. – Gravid uterus with cervix

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Caesarean Section Data

Group/ Dose

(%)

No. of Corpus Luteum

No. of Implantation

No. of Embryo Foetal Death

Implantation Loss Rate (%)

Resorption Rate (%)

Embryo Foetal Mortality

(%)

No. of Live Foetuses

Sex Ratio

(%)

Resorption

Dead Foetuses

Pre-

Post-

Early

Late

Sex

Total

Early

Late

Male

Female

G1: 0

Mean

15.4

14.7

0.5

0.0

0.0

5.1

3.6

3.6

0.0

3.6

7.0

7.2

14.2

49.8

S.D

2.0

2.3

0.8

0.0

0.0

7.3

5.8

5.8

0.0

5.8

1.8

2.4

2.6

11.4

N

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

14.4

13.7

0.2

0.0

0.0

4.8

6.3

6.3

0.0

6.3

3.9

6.6

13.5

51.2

S.D

3.6

3.7

0.5

0.0

0.0

8.9

22.9

22.9

0.0

22.9

2.6

2.6

3.9

11.5

N

19

19

19

19

19

19

19

19

19

19

19

19

19

19

G3: 0.5

Mean

14.8

14.5

0.7

0.0

0.1

2.1

5.5

5.1

0.0

5.5

7.0

6.7

13.7

51.0

S.D

2.1

1.8

0.8

0.0

0.2

5.3

6.0

5.6

0.0

6.0

1.9

1.6

2.2

10.3

N

17

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

15.0

14.7

0.6

0.0

0.0

2.4

4.0

4.0

0.0.

4.0

8.0

6.1

14.1

56.4

S.D

1.2

1.4

0.7

0.0

0.0

4.1

4.6

4.6

0.0

4.6

2.3

2.1

1.4

15.2

N

20

20

20

20

20

20

20

20

20

20

20

20

20

20

Pre-implantation loss rate (%) = ((No. of corpora lutea – no. of implantation sites) / No. of corpora lutea) x 100

Post-implantation loss rate (%) = ((No. of implantations – No. of live foetuses) / No. of implantations) x 100

Early resorption rate (%) = (No. of early resorptions / No. of implantations) x 100

Late resorption rate (%) = (No. of late resorptions / No. of implantations) x 100

Embryo foetal mortality rate (%) = (No. of resorptions + No. of dead foetuses / No. of implantations) x 100

Sex ration (%) = (No. of live male foetuses / No. of total live foetuses) x 100

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean External Findings of Foetuses and Placentas

Group/ Dose

(%)

No. of Dams

 

Foetuses

Placentas

Abnormal

Shared

Large

Small

G1:0

19

Total

0.00 (0)

0.00 (0)

 

0.00 (0)

Incidence (%)

0.00

0.00

 

0.00

G2: 0.25

18

Total

0.00 (0)

0.13 (2)

0.00 (0)

0.00 (0)

Incidence (%)

0.00

0.72

0.00

0.00

G3: 0.5

17

Total

0.00 (0)

0.00 (0)

0.00 (0)

0.08 (1)

Incidence (%)

0.00

0.00

0.00

0.47

G4: 1.0

20

Total

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

0.00

0.00

0.00

0.00

( ): No. of found foetuses

Value = No of found foetuses / No. of foetuses per litter

Incidence (%) = (Total value / No. of dams) x 100

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Foetal Body Weights and Placental Weights (g)

Group/ Dose

(%)

Body Weights

Placenta Weights

Total

Male

Female

Total

Male

Female

G1: 0

Mean

3.90

4.00

3.80

0.45

0.46

0.44

S.D

0.25

0.27

0.24

0.04

0.05

0.05

N

19

19

19

19

19

19

G2: 0.25

Mean

3.94

4.05

3.84

0.44

0.45

0.44

S.D

0.52

0.54

0.52

0.04

0.03

0.05

N

18

18

18

18

18

18

G3: 0.5

Mean

3.95

4.04

3.85

0.47

0.47

0.46

S.D

0.27

0.33

0.24

0.06

0.07

0.06

N

17

17

17

17

17

17

G4: 1.0

Mean

3.97

4.06

3.87

0.46

0.47

0.45

S.D

0.19

0.20

0.20

0.03

0.03

0.03

N

20

20

20

20

20

20

N: No. of dams.

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Anogenital Distance (mm)

Group/ Dose

(%)

Anogenital Distance

Male

Female

G1: 0

Mean

1.26

0.58

S.D

0.05

0.05

N

19

19

G2: 0.25

Mean

1.26

0.59

S.D

0.07

0.06

N

18

18

G3: 0.5

Mean

1.25

0.58

S.D

0.05

0.05

N

17

17

G4: 1.0

Mean

1.26

0.60

S.D

0.07

0.05

N

20

20

N: No. of dams.

AGD index = AGD/^3√(body weight).

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Visceral Findings of Foetuses

Group / Dose

(%)

No. of Dams

 

Variation

Ureter

Kidneys

Umbilical Artery

Dilated

Convoluted

Dilated Renal Pelvis

Malpositioned

G1: 0

19

Total

2.31 (6)

4.54 (32)

0.00 (0)

0.13 (1)

Incidence (%)

12.16

23.89

0.0

0.68

G2: 0.25

18

Total

1.10 (8)

3.32 (24)

0.00 (0)

0.13 (1)

Incidence (%)

6.11

18.44

0.00

0.72

G3: 0.5

17

Total

1.28 (8)

4.40 (30)

0.28 (2)

0.27 (2)

Incidence (%)

7.53

25.88

1.65

1.59

G4: 1.0

16

Total

1.32 (9)

4.00 (28)

0.00 (0)

0.26 (2)

Incidence (%)

6.60

20.00

0.00

1.30

( ): No. of found foetuses.

Total value = sum (No. of found foetuses / No. of foetuses per litter).

Incidence (%) = (Total value / No. of dams) x 100.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Skeletal Findings of Foetuses

Group / Dose

(%)

No. of Dams

 

Variation

Skull

Rib

Parietal Bone

Supraoccipital Bone

Interparietal

Short

Bent

Wavy

Lumbar Rib

Incomplete Ossification

Incomplete Ossification

Biparite Ossification

Incomplete Ossification

G1: 0

19

Total

0.29 (2)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

1.56 (10)

Incidence (%)

1.53

0.00

0.00

0.00

0.00

0.00

0.00

8.21

G2: 0.25

18

Total

0.00 (0)

0.00 (0)

0.15 (1)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

3.12 (20)

Incidence (%)

0.00

0.00

0.83

0.00

0.00

0.00

0.00

17.33

G3: 0.5

17

Total

0.63 (4)

0.15 (1)

0.00 (0)

0.00 (0)

0.35 (2)

0.17 (1)

0.00 (0)

2.15 (15)

Incidence (%)

3.71

0.88

0.00

0.00

2.06

1.00

0.00

12.65

G4: 1.0

16

Total

0.57 (4)

0.00 (0)

0.00 (0)

0.25 (2)

0.00 (0)

0.00 (0)

0.00 (0)

2.36 (16)

Incidence (%)

2.85

0.00

0.00

1.25

0.00

0.00

0.00

11.80

 

Group / Dose

(%)

No. of Dams

 

Variation

Vertebra

Pelvic Girdle

Thoracic

Sternebra

Ischiu

Pubis

Dumbbell Shaped

Bipartite Ossification

Bipartite Ossification

Dumbbell Shaped

Incomplete ossification

Incomplete ossification

G1: 0

19

Total

0.20 (1)

0.59 (4)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

1.05

3.11

0.00

0.00

0.00

0.00

G2: 0.25

18

Total

0.30 (2)

1.07 (7)

0.15 (1)

0.00 (0)

0.25 (2)

0.54 (4)

Incidence (%)

1.67

5.94

0.83

0.00

1.39

3.00

G3: 0.5

17

Total

0.33 (2)

0.47 (3)

0.00 (0)

0.00 (0)

0.25 (2)

0.58 (4)

Incidence (%)

1.94

2.76

0.00

0.00

1.47

3.41

G4: 1.0

16

Total

0.15 (1)

0.32 (2)

0.00 (0)

0.00 (0)

0.00 (0)

0.00 (0)

Incidence (%)

0.75

1.60

0.00

0.00

0.00

0.00

( ): No. of found foetuses.

Total value = sum (No. of found foetuses / No. of foetuses per litter).

Incidence (%) = (Total value / No. of dams) x 100.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Mean Ossification Status of Foetuses

Group / Dose

(%)

No. of Foetuses

OSSB

Pairs of Ribs

Sternebra

Sacro-caudal vertebra

Fore Limb

Hind Limb

Mc

Pp

Mp

Dp

Mt

Pp

Mp

Dp

G1: 0

Mean

6.9

1.9

13.0

5.6

7.9

7.5

0.4

0.0

0.0

8.0

0.0

0.0

0.0

S.D

1.3

0.1

0.1

0.4

0.5

0.6

0.9

0.0

0.0

0.0

0.0

0.0

0.0

N

19

19

19

19

19

19

19

19

19

19

19

19

19

G2: 0.25

Mean

6.9

1.8

13.1

5.6

8.0

7.5

0.7

0.0

0.0

8.1

0.1

0.0

0.0

S.D

1.1

0.2

0.1

0.4

0.7

0.6

1.5

0.0

0.0

0.4

v

0.0

0.0

N

18

18

18

18

18

18

18

18

18

18

18

18

18

G3: 0.5

Mean

6.6

1.9

13.0

5.5

7.9

7.5

0.5

0.0

0.0

8.0

0.0

0.0

0.0

S.D

1.1

0.1

0.1

0.4

0.4

0.5

0.9

0.0

0.0

0.0

0.0

0.0

0.0

N

17

17

17

17

17

17

17

17

17

17

17

17

17

G4: 1.0

Mean

6.9

1.9

13.1

5.6

8.0

7.5

0.4

0.0

0.0

8.0

0.0

0.0

0.0

S.D

0.8

0.2

0.1

0.4

0.4

0.5

0.5

0.0

0.0

0.0

0.0

0.0

0.0

N

20

20

20

20

20

20

20

20

20

20

20

20

20

N: No. of dams.

OSSB: Ossified step of supraoccipital bone.

Mc: Metacarpal, Pp: Proximal phalanx, Mp: Middle phalanx, DP: Distal phalanx, Mt: Metatarsal.

Significantly different from control by Dunnett's t-test: * p<0.05, ** p><0.01. Significantly different from control by DSCF: # p><0.05, ## p><0.05, ** p<0.01.

Significantly different from control by DSCF: # p<0.05, ## p <0.01.

 

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).
Executive summary:

A prenatal developmental toxicity study was carried out in rats in accordance with the standardised guideline OECD 414 and NIER Notification No. 2020-46 under GLP conditions.

The purpose of this study was to investigate the effects of the test material on pregnant females and prenatal development when administered orally (via the diet) to pregnant female Sprague-Dawley rats (20 animals per group) from implantation to the day prior to scheduled caesarean section (from Gestation Day (GD) 5 to GD 19, total 15 days). The administration was carried out at doses of 0 (control), 0.25, 0.5, and 1.0 % (0, 221.3, 454.6 and 925.1 mg/kg/day, respectively).

Evaluated parameters included clinical signs, body weight, food consumption, thyroid hormone analysis, organ weights, gross post-mortem examinations, histopathology, caesarean section, external examination of foetuses and placentas, measurement of body weights of live foetuses and placental weights, anogenital distance (AGD), and foetal visceral and skeletal examination.

No deaths related to the test material occurred in the treatment groups during the study period.

No test material-related changes were observed in the clinical signs, body weight, food consumption, thyroid hormone levels, organ weights, caesarean section, gross pathology and histopathology at any dose tested.

No test material-related changes were observed in the external examination of foetuses and placentas, foetal body weight and placental weight, AGD index, and visceral and skeletal examination at any dose tested.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test substance for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
30 March 2021 to 28 June 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl:CD(SD), SPF
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males were 10 weeks old and females were 9 weeks old at receipt and at the start of mating. Females were 10 – 12 weeks old at the start of administration.
- Weight at study initiation: Males were 335.8 – 362.7 g and females were 198.9 – 226.7 g at receipt. Females were 238.9 – 280.1 g at the start of administration.
- Fasting period before study: No
- Housing: Animals were housed in stainless wire mesh cages, 260 W × 350 D × 210 H (mm). Then from GD 16 to necropsy animals were housed in polycarbonate cages, 260 W × 420 D × 180 H (mm). One – two animals were housed per cage during the quarantine acclimation period and females were housed individually during the gestation period.
- Diet: Powder feed rodent chow was placed in feeders and provided ad libitum.
- Water: Public tap water was filtered and irradiated by a UV water steriliser and then provided ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0 - 24.3 °C.
- Humidity (%): 45.6 – 63.8 % (relative).
- Air changes (per hr): 10 – 15 clean, fresh, filtered air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle (7 AM to 7 PM via automated timer), 150 - 300 Lux.

IN-LIFE DATES:
From: 25 March 2021
To: 09 August 2021
Route of administration:
oral: feed
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test material was weighed and placed in a container. The required amount of vehicle was added and mixed using a vortex mixer until dissolved.
The required amount of powder feed except for the amount of the test material was weighed. The required amount of the test material formulation and a small amount of powder feed were mixed in a container. And then, the mixture was placed in a ball mill and residual powder feed was added and mixed using the ball mill for approximately 5 – 10 minutes to yield the desired concentration.
The required amounts of corn oil and powder feed were weighed on an electronic balance and mixed using the ball mill for approximately 5 – 10 minutes for the control group.

DIET PREPARATION
- Storage temperature of food: The dosing feed was stored under refrigeration condition within 14 days and used at room temperature (1 – 30 °C) for 6 days.

VEHICLE
- Concentration in vehicle: 10 mL of vehicle for 1 kg of powder feed containing test substance
Analytical verification of doses or concentrations:
no
Remarks:
Analysis for the concentration of dosing formulations was not performed.
Details on analytical verification of doses or concentrations:
The homogeneity and stability at room temperature (1 – 30 °C) for 3 and 6 days and under refrigeration for 8 and 14 days of the dosing formulations comprising the dose levels of 0.04 and 1.5 % were confirmed.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1. During the mating period, virgin female rats were cohabitated with male breeder rats (one male rat per one female rat) in the home cages housing sexually mature breeder males. Female rats without evidence of mating were separated from the male rats in the morning and individually housed until that afternoon. Then, the male and female rats were cohabitated again in the afternoon.
- Length of cohabitation: The mating period consisted of a maximum of 5 days.
- Proof of pregnancy: Confirmation of pregnancy was conducted the next morning. Mating was evaluated daily during the cohabitation period. The day when female rats were observed with spermatozoa in a smear of the vaginal contents and/or the presence of a copulatory plug was observed in situ was considered to be GD 0.
Duration of treatment / exposure:
Gestation Day (GD) 5 to GD 19.
Frequency of treatment:
Mixed test substance formulation and powder feed were placed in feeders and provided ad libitum.
Duration of test:
GD 5 to GD 19, total 15 days.
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
0 %
Dose / conc.:
149.4 mg/kg bw/day (nominal)
Remarks:
0.17 %
Dose / conc.:
450.7 mg/kg bw/day (nominal)
Remarks:
0.5 %
Dose / conc.:
1 446.7 mg/kg bw/day (nominal)
Remarks:
1.5 %
No. of animals per sex per dose:
6 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: As a result of a 2-week dose range finding study (for the repeated dose endpoint) no test material-related effect was observed at 1.5 %. Therefore, 1.5 % was selected as the high dose level in this study. The mid and low doses were selected at 0.5 % and 0.17 %, respectively. The control animals were received the standard basal powder feed with corn oil (vehicle).
- Rationale for animal assignment: On the day following mating, copulated females with body weights close to the mean body weight were selected and assigned to 4 groups (6 animals/ group). Selected animals were randomly assigned to achieve an even distribution of mean body weight of each group.
Maternal examinations:
The parameters listed below were evaluated for all copulated females. However, pregnancy was only finally confirmed at the time of caesarean section.

CAGE SIDE AND CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily for clinical signs and twice daily for mortality, moribundity, abortion and premature birth during the observation period.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on GDs 0, 3, 5, 8, 11, 14, 17, 19 and 20 (the day of necropsy) using an automatic animal balance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule for examinations: For the determination of food consumption for individual animals, feed of measured quantity was offered on GDs 0, 2, 4, 7, 10, 13, 16 and 18, and the amount of feed left was measured on the respective following day.
- Food consumption: Individual food consumption data was determined by subtracting the amount of feed left from the given quantity.
- Test material intake: The test substance intake was calculated as follows and designated by mg/kg/day:
Test material intake (mg/kg/day) = [Food consumption (mg/day) × Concentration (%)] / Body weight (kg)

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20. All animals were sacrificed by exsanguination from the abdominal aorta under isoflurane anaesthesia on the day of necropsy.
- Gross pathology examinations: Complete gross post-mortem examinations were performed on all animals including the external surface and internal organs. All grossly visible abnormalities were recorded.
- Organ weights: The following organs of all terminal sacrifice animals were weighed individually and the relative ratio of organ weight to terminal body weight of dams (excluding gravid uterus with cervix weight) was calculated: Gravid uterus with cervix, thyroid gland with parathyroid gland (weighed paired and after fixation) and liver.
- Histopathology: At necropsy, the following organs and tissues from all pregnant animals were harvested and preserved in 10 % neutral buffered formalin: Brain, pituitary gland, ovary, uterus with cervix, liver and thyroid gland.
Histopathological examination of the samples of the following organs and tissues from all pregnant animals was performed: Liver and thyroid gland.
Ovaries and uterine content:
The ovaries and uterine content were examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

At necropsy, the ovaries and uterus of all females were removed and implantations, early and late resorptions and dead/ live foetuses were examined; the number of corpora lutea and implantation sites were counted. The pre- & post-implantation loss rates, early & late resorptions rates and foetal mortality rate were calculated.
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: No
- Skeletal examinations: No
- Head examinations: No
- Anogenital distance of all pups: Yes

- Sex ratio: The sex of each foetus was determined by comparing the distance between the anus and the genital tubercle (short for females, long for males).

- External examination of foetuses and observation of placenta: All foetuses were examined for external findings including the eyes, ears, nose, mouth, palate, absence of limbs and tail, position and size, shape etc. In addition, the shape and colour of the placenta were observed.

- Body weights of live foetuses and placenta weights: Live foetuses removed from the uterus were numbered sequentially from the left ovary to the right ovary on the dorsal surface with an indelible pen. The weights of surviving foetuses and placental weights were recorded. Foetal body weights and placental weights were based on the average value of the foetuses of the same litter. All live foetuses were euthanised by hypothermia.
Statistics:
Statistical analysis was performed for the body weights, food consumption, organ weights, number of corpora lutea, number of implantations, pre-/ post-implantation loss rates, body weights and placenta weights of live foetuses and number of live foetuses using the SAS program (version 9.4, SAS Institute Inc., U.S.A.).
The data was analysed utilising Bartlett’s test for homogeneity of variance (significance level: 0.05). One-way analysis of variance (ANOVA) was employed on homogeneous data (significance level: 0.05). The Kruskal-Wallis test was employed on heterogeneous data (significance level: 0.05).
The results of other data types such as malformation and anomalies from the external findings, early resorption rate, late resorption rate and foetal mortality rate were analysed utilising the Kruskal-Wallis test (significance level: 0.05).
Indices:
- Pre-implantation loss rate (%) = [(No. of corpora lutea – No. of implantations) / No. of corpora lutea] × 100
- Post-implantation loss rate (%) = [(No. of implantations – No. of live foetuses ) / No. of implantations] × 100
- Early resorptions (%) = (No. of early resorptions / No. of implantations) × 100
- Late resorptions (%) = (No. of late resorptions / No. of implantations) × 100
- Foetal mortality (%) = (No. of dead foetuses / No. of implantations) × 100
- Sex ratio (%) = (No. of live male foetuses / No. of total live foetuses) × 100
Clinical signs:
no effects observed
Description (incidence and severity):
There were no abnormalities or clinical signs in the animals in the control group, 0.17, 0.5 and 1.5 % groups during the observation period.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no deaths in the animals in the control group, 0.17, 0.5 and 1.5 % groups during the observation period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No test material-related effects on the body weights and body weight gains were observed in the 0.17, 0.5 and 1.5 % groups during the study period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No test material-related effects on food consumption and relative food consumption were observed in the 0.17, 0.5 and 1.5 % groups during the study period.
The mean total test material intakes in the 0.17, 0.5 and 1.5 % groups were 149.4, 450.7 and 1 446.7 mg/kg/day, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There was no significant difference in the absolute and relative organ weights between the control group and test material dosing groups. No test material-related change in placenta weights were observed in the 0.17, 0.5 and 1.5 % groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
At necropsy, no noticeable findings were noted in any animals. No test material-related effect was observed in the 0.17, 0.5 and 1.5 % groups in examination of placentas.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination did not reveal treatment-related changes in any animals. No other remarkable microscopic findings were observed in any animals.
Histopathological findings: neoplastic:
not specified
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
No test material-related changes in caesarean section parameters were observed in the 0.17, 0.5 and 1.5 % groups.
Dose descriptor:
NOAEL
Effect level:
1 446.7 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other:
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
No test material-related change in foetal body weights were observed in the 0.17, 0.5 and 1.5 % groups.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
not specified
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No test material-related effect was observed in the 0.17, 0.5 and 1.5 % groups in external examination of foetuses. Other changes observed, including a foetus with subcutaneous oedema, small foetus, shared or small placentas in the 0.17 and 1.5 % groups were observed without dose-dependency. Therefore, these changes were considered to be not test material-related.
Skeletal malformations:
not examined
Visceral malformations:
not examined
Dose descriptor:
NOAEL
Effect level:
1 446.7 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Abnormalities:
no effects observed
Developmental effects observed:
no

Summary of Clinical Signs






































































































































Group/ Dose


(%)



No. of Animals



Clinical Sign



Gestation Day



0



1



2



3



4



5



6



7



8



9



10



11



12



13



14



15



16



17



18



19



G1: 0



6



NOA



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G2: 0.17



6



NOA



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G3: 0.5



6



NOA



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G4: 1.5



6



NOA



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



6



NOA: No Observable Abnormality.


 


Mean Body Weights (g)









































































































































































Group/ Dose


(%)



Gestation Day



0



3



5



8



11



14



17



19



20



G1: 0



Mean



234.5



253.8



263.4



274.5



292.2



316.1



344.6



390.2



413.3



S.D.



8.2



8.4



8.3



8.1



9.1



10.4



13.1



8.8



7.2



N



6



6



6



6



6



6



6



6



6



G2: 0.17



Mean



235.4



253.1



262.0



274.6



293.9



311.0



338.6



337.8



400.9



S.D.



7.5



10.5



10.1



12.7



13.7



17.6



23.1



26.2



28.6



N



6



6



6



6



6



6



6



6



6



G3: 0.5



Mean



234.2



249.5



259.4



271.6



291.0



310.5



341.1



378.0



401.9



S.D.



12.9



13.7



14.2



14.3



16.9



17.7



16.4



17.9



20.8



N



6



6



6



6



6



6



6



6



6



G4: 1.5



Mean



234.6



252.2



266.1



273.6



293.0



310.4



337.7



383.8



411.0



S.D.



6.8



7.9



9.5



11.7



13.6



14.2



22.1



20.5



24.3



N



6



6



6



6



6



6



6



6



6



 


Mean Body Weight Gains (g)




























































































































































Group/ Dose


(%)



Gestation Day



3



5



8



11



14



17



19



20



G1: 0



Mean



19.3



9.7



11.1



17.6



24.0



28.5



45.6



23.1



S.D.



7.3



2.9



3.7



2.7



5.4



11.3



8.6



4.0



N



6



6



6



6



6



6



6



6



G2: 0.17



Mean



17.7



8.9



12.6



19.4



17.1



27.7



39.2



23.1



S.D.



6.2



2.3



3.4



2.9



5.1



9.9



10.6



7.7



N



6



6



6



6



6



6



6



6



G3: 0.5



Mean



15.3



10.0



12.2



19.4



19.5



30.6



36.9



23.9



S.D.



5.3



3.0



4.2



3.5



1.9



4.5



3.8



3.7



N



6



6



6



6



6



6



6



6



G4: 1.5



Mean



17.6



13.9



7.5



19.4



17.4



27.3



46.1



27.2



S.D.



5.8



4.4



6.9



8.1



7.2



15.6



16.8



4.7



N



6



6



6



6



6



6



6



6



 


Mean Food Consumption (g/day)




























































































































































Group/ Dose


(%)



Gestation Day



1



3



5



8



11



14



17



19



G1: 0



Mean



24.2



24.0



25.6



27.0



24.8



27.6



29.9



28.8



S.D.



3.8



2.3



1.5



2.4



1.8



2.6



2.5



1.4



N



5^



6



6



6



6



6



6



6



G2: 0.17



Mean



22.4



24.0



26.2



24.4



25.7



26.8



28.2



29.5



S.D.



4.4



2.6



2.0



3.1



2.7



3.3



2.6



3.1



N



6



6



6



5^



6



6



6



6



G3: 0.5



Mean



22.4



23.5



25.2



25.6



24.8



26.8



27.5



31.4



S.D.



4.1



2.9



3.2



3.2



2.5



3.3



3.9



3.8



N



6



6



6



6



6



6



6



6



G4: 1.5



Mean



22.4



24.5



25.4



24.9



25.3



25.7



29.0



30.7



S.D.



2.1



2.4



2.8



3.3



2.4



2.7



4.0



1.3



N



6



6



6



6



6



6



6



6



^: The animal that abandoned most of the feed from the feeder was excluded from the data.


 


Mean Relative Food Consumption (g/kg body weight/day)




























































































































































Group/ Dose


(%)



Gestation Day



1



3



5



8



11



14



17



19



G1: 0



Mean



103.8



94.3



97.1



98.4



84.7



87.0



86.6



73.8



S.D.



160



6.2



4.2



9.2



5.4



6.2



5.6



4.4



N



5^



6



6



6



6



6



6



6



G2: 0.17



Mean



94.9



102.1



100.1



89.0



87.3



85.9



83.5



78.0



S.D.



17.2



10.5



8.6



7.5



6.3



6.5



6.9



5.2



N



6



6



6



5^



6



6



6



6



G3: 0.5



Mean



93.5



94.1



97.1



93.9



85.1



86.1



80.5



83.0



S.D.



13.8



6.8



10.4



8.6



4.2



6.0



10.1



8.5



N



6



6



6



6



6



6



6



6



G4: 1.5



Mean



95.3



97.1



95.2



91.2



86.3



82.7



85.5



80.2



S.D.



8.8



8.1



7.1



12.6



5.5



6.7



66



6.5



N



6



6



6



6



6



6



6



6



Dosing period: Gestation Day 5 – 19.


^: The animal that abandoned most of the feed from the feeder was excluded from the data.


 


Mean Test Material Intake (mg/kg/day)
















































































































Group/ Dose


(%)



Gestation Day



Total Mean



5



8



11



14



17



19



G2: 0.17



Mean



166.3



146.5



145.2



145.6



137.5



130.9



149.4



S.D.



13.0



20.2



15.5



20.2



15.4



18.3



21.5



N



6



5^



6



6



6



6



6



G3: 0.5



Mean



478.2



479.2



441.0



454.2



415.5



436.0



450.7



S.D.



60.6



65.0



47.9



67.7



66.6



47.7



53.1



N



6



6



6



6



6



6



6



G4: 1.5



Mean



1 427.0



1 547.3



1 443.1



1 416.6



1 438.3



1 407.9



1 446.7



S.D.



180.3



142.8



79.5



156.3



177.2



79.4



72.9



N



6



6



6



6



6



6



6



Dosing period: Gestation Day 5 – 19.


Total mean: The mean values during the dosing period.


^: The animal that abandoned most of the feed from the feeder was excluded from the data.


 


Mean Absolute Organ Weights (g)





































































































Group/ Dose


(%)



B.W.



Liver



Gravid Uterus with Cervix



Thyroid Gland with Parathyroid Gland



G1: 0



Mean



413.3



16.22



85.05



0.0211



S.D.



7.2



0.98



5.68



0.0058



N



6



6



6



6



G2: 0.17



Mean



400.9



16.18



75.96



0.0209



S.D.



28.6



0.96



11.67



0.0022



N



6



6



6



6



G3: 0.5



Mean



401.9



15.88



84.02



0.0210



S.D.



20.8



1.61



4.44



0.0034



N



6



6



6



6



G4: 1.5



Mean



411.0



16.68



86.33



0.0246



S.D.



24.3



1.60



11.23



0.0054



N



6



6



6



6



 


Relative Mean Organ Weights (g/100 g body weight)





































































































Group/ Dose


(%)



Correct B.W.


(g)



Liver



Gravid Uterus with Cervix



Thyroid Gland with Parathyroid Gland



G1: 0



Mean



328.2



4.9421



25.9820



0.0064



S.D.



11.3



0.2674



2.5463



0.0017



N



6



6



6



6



G2: 0.17



Mean



324.9



4.9800



23.2879



0.0065



S.D.



17.8



0.0988



2.5654



0.0008



N



6



6



6



6



G3: 0.5



Mean



317.9



4.9848



26.4526



0.0067



S.D.



17.4



0.2502



1.0496



0.0012



N



6



6



6



6



G4: 1.5



Mean



234.7



4.2096



26.5855



0.0076



S.D.



17.3



0.5894



3.9060



0.0016



N



6



6



6



6



Correct B.W. = Termina B.W. – Gravid uterus.


 


Mean Caesarean Section Data

















































































































































































































































Group/ Dose


(%)



No. of Corpus Lueum



No. of Implantations



No. of Embry Foetal Death



Implantation Loss Rate (%)



Resorption Rate (%)



Foetal Mortality


(%)



No. of Live Foetuses



Sex Ration


(%)



Resorption



Dead Foetuses



Pre-



Post-



Early



Late



Sex



Total



Early



Late



Male



Female



G1: 0



Mean



15.3



14.8



0.7



0.0



0.0



3.0



4.4



4.4



0.0



0.0



6.7



7.5



14.2



47.2



S.D.



1.5



1.0



0.5



0.0



0.0



4.9



3.4



3.4



0.0



0.0



2.3



2.4



0.8



16.3



N



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G2: 0.17



Mean



14.5



13.3



0.5



0.0



0.0



8.0



3.6



3.6



0.0



0.0



7.7



5.2



12.8



59.1



S.D.



2.1



2.3



0.8



0.0



0.0



9.2



5.8



5.8



0.0



0.0



2.7



2.2



2.3



18.0



N



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G3: 0.5



Mean



14.7



14.5



0.5



0.0



0.0



1.1



3.4



3.3



0.0



0.0



8.3



5.7



14.0



59.7



S.D.



1.0



0.8



0.5



0.0



0.0



2.6



3.7



3.7



0.0



0.0



1.0



1.4



0.6



8.3



N



6



6



6



6



6



6



6



6



6



6



6



6



6



6



G4: 1.5



Mean



15.7



15.0



0.5



0.2



0.0



4.8



4.1



3.0



1.0



0.0



7.3



7.0



14.3



50.1



S.D.



1.6



2.6



0.8



0.5



0.0



9.3



5.0



3.3



2.6



0.0



3.1



2.2



2.3



16.3



N



6



6



6



6



6



6



6



6



6



6



6



6



6



6



Pre-imp. Loss rate (%) = ((No. of corpora lutea – No. of implantations) / No. of corpora lutea) x 100.


Post-imp. Loss rate (%) = ((No. of implantations – No. of live pups) / No. of implantations) x 100.


Early resorption rate (%) = (No of early resorptions / No. of implantation) x 100.


Late resorption rate (%) = (No of late resorptions / No. of implantation) x 100.


Foetal mortality *%) = (No. of dead foetuses / No. of implantation) x 100.


Sex ratio (%) = No. of live male foetuses / No. of total live foetuses x 100


 


Mean Foetal Body Weights and Placenta Weights



































































































































Group/ Dose


(%)



Body Weights



Placenta Weights



Total



Male



Female



Total



Male



Female



G1: 0



Mean



3.91



4.05



3.76



0.48



0.48



0.48



S.D.



0.17



0.16



0.20



0.04



0.05



0.04



N



6



6



6



6



6



6



G2: 0.17



Mean



3.77



3.81



3.72



0.48



0.47



0.49



S.D.



0.26



0.23



0.36



0.05



0.05



0.04



N



6



6



6



6



6



6



G3: 0.5



Mean



3.88



4.02



3.73



0.46



0.47



0.45



S.D.



0.07



0.10



0.07



0.04



0.05



0.02



N



6



6



6



6



6



6



G4: 1.5



Mean



3.87



3.95



3.80



0.51



0.51



0.51



S.D.



0.16



0.19



0.14



0.08



0.05



0.10



N



6



6



6



6



6



6



N: No. of dams.


 


Mean External Findings of Foetuses and Placentas



















































































Group/ Dose


(%)



No. of Dams



 



Foetuses



Placentas



Subcutaneous Oedema (Generalised), General



Small



Shared



Small



G1: 0



6



Total



0.00 (0)



0.00 (0)



0.00 (0)



0.00 (0)



Incidence (%)



0.00



0.00



0.00



0.00



G2: 0.17



6



Total



0.10 (1)



0.07 (1)



0.32 (4)



0.10 (1)



Incidence (%)



1.67



1.11



5.25



1.67



G3: 0.5



6



Total



0.00 (0)



0.00 (0)



0.00 (0)



0.00 (0)



Incidence (%)



0.00



0.00



0.00



0.00



G4: 1.5



6



Total



0.00 (0)



0.00 (0)



0.13 (2)



0.00 (0)



Incidence (%)



0.00



0.00



2.08



0.00



Total value = Sum (No. of found foetuses / No. of foetuses per litter).


Incidence (%) = (Total value / No. of dams) x 100.


( ): No. of found foetuses


 

Conclusions:
Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.5 % (1 446.7 mg/kg/day).
Executive summary:

A dose range-finding study to determine the dose levels for a prenatal developmental toxicity study was carried out in rats with reference to the standardised guideline OECD 414 under GLP conditions.

The purpose of this study was to determine the dose range for the prenatal developmental toxicity study by evaluating the effects on dams and embryo-foetal development when administered orally (in the diet) to pregnant Sprague-Dawley (SD) rats from implantation to the day prior to scheduled caesarean section (from Gestation Day (GD) 5 to GD 19, total 15 days). The administration was conducted at doses of 0 (vehicle control), 0.17, 0.5, and 1.5 % (0, 149.4, 450.7 and 1 446.7 mg/kg/day, respectively).

Evaluated parameters included clinical signs, body weight, food consumption, organ weights, gross pathology, histopathology, caesarean section, external examination of foetuses and placentas, measurement of body weights of live foetuses and placenta weights.

No clinical signs or deaths related to the test substance occurred in the treatment groups during the study period. No test material-related changes were observed in the body weight, food consumption, organ weights, and caesarean section at any dose tested.

No test material-related changes were observed in the external examination of foetuses and placentas, foetal body weight and placenta weight.

Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.5 % (1 446.7 mg/kg/day).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
925.1 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Single study on the target substance, supported by a dose range finder study, conducted according to standardised guidelines and in compliance with GLP in an appropriate test species.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
710 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The overall quality of the database is good. Two studies on similar substance were available, both were assigned a reliability score of 2 in accordance with the principles for assessing data quality outlined in Klimisch (1997) as they were both reported to a high standard, and the methods followed were in basic compliance with the current standardised guidelines. The data is sufficient to address effects on development on the basis of weight of evidence.
Additional information

Oral Route Key Study: Hamm (2022)


A prenatal developmental toxicity study was carried out in rats in accordance with the standardised guideline OECD 414 and NIER Notification No. 2020-46 under GLP conditions.  The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


The purpose of this study was to investigate the effects of the test material on pregnant females and prenatal development when administered orally (via the diet) to pregnant female Sprague-Dawley rats (20 animals per group) from implantation to the day prior to scheduled caesarean section (from Gestation Day (GD) 5 to GD 19, total 15 days). The administration was carried out at doses of 0 (control), 0.25, 0.5, and 1.0 % (0, 221.3, 454.6 and 925.1 mg/kg/day, respectively).


Evaluated parameters included clinical signs, body weight, food consumption, thyroid hormone analysis, organ weights, gross post-mortem examinations, histopathology, caesarean section, external examination of foetuses and placentas, measurement of body weights of live foetuses and placental weights, anogenital distance (AGD), and foetal visceral and skeletal examination.


No deaths related to the test material occurred in the treatment groups during the study period.


No test material-related changes were observed in the clinical signs, body weight, food consumption, thyroid hormone levels, organ weights, caesarean section, gross pathology and histopathology at any dose tested.


No test material-related changes were observed in the external examination of foetuses and placentas, foetal body weight and placental weight, AGD index, and visceral and skeletal examination at any dose tested.


Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test substance for both pregnant rats and embryofoetal development are considered to be at 1.0 % (925.1 mg/kg/day).


 


Oral Route Supporting Study: Hamm (2021)


A dose range-finding study to determine the dose levels for a prenatal developmental toxicity study was carried out in rats with reference to the standardised guideline OECD 414 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).


The purpose of this study was to determine the dose range for the prenatal developmental toxicity study by evaluating the effects on dams and embryo-foetal development when administered orally (in the diet) to pregnant Sprague-Dawley (SD) rats from implantation to the day prior to scheduled caesarean section (from Gestation Day (GD) 5 to GD 19, total 15 days). The administration was conducted at doses of 0 (vehicle control), 0.17, 0.5, and 1.5 % (0, 149.4, 450.7 and 1 446.7 mg/kg/day, respectively).


Evaluated parameters included clinical signs, body weight, food consumption, organ weights, gross pathology, histopathology, caesarean section, external examination of foetuses and placentas, measurement of body weights of live foetuses and placenta weights.


No clinical signs or deaths related to the test substance occurred in the treatment groups during the study period. No test material-related changes were observed in the body weight, food consumption, organ weights, and caesarean section at any dose tested.


No test material-related changes were observed in the external examination of foetuses and placentas, foetal body weight and placenta weight.


Under the conditions of this study, the no-observed-adverse-effect level (NOAEL) of the test material for both pregnant rats and embryofoetal development are considered to be at 1.5 % (1 446.7 mg/kg/day).


 


Dermal Route WoE Read-Across Source: Palmer (1986)


Doses of 1.40, 0.43 and 0.14 mL/kg/day were administered occlusively to the skin to Crl rats between days 6 to 15 of gestation. Maternal animals were observed for signs of toxicity and local irritation. On gestation day 20, the animals were sacrificed and developmental parameters were assessed in the pups in utero. In addition to the developmental toxicity study, an absorption study was included to give a brief picture on the behaviour of the substance when dosed dermally. For this purpose 3 animals from the low and high dose groups received a radiolabelled dose on the final day of treatment.


 The test material was found to be rapidly and extensively absorbed in the pilot study.


 Both maternal and embryo-foetal toxicity was observed at a dose of 1.40 mL/kg bw/day (1428 mg/kg bw/day). Given the physical chemical properties and ability to produce narcosis the embryo-foetal toxic effects were considered to be independent of the maternal toxicity, but likely to stem from the same physical-chemical mechanisms of toxicity. The 0.43 mL/kg bw/day (439 mg/kg bw/day) dose group was considered to be close to the threshold of maternal toxicity, but is considered the NOAEL for maternal toxicity in this study. An enhancement of the incidence of reversible variations and anomalies were recorded in the individual foetuses, litter values however were not affected. No malformations were recorded at this dose level.


 Under the conditions of the test, the NOAEL for maternal toxicity, embryo- and developmental toxicity was 0.43 mL/kg bw/day (439 mg/kg bw/day). The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Dermal Route WoE Read-Across Source: Hoberman (1988)


The developmental toxicity of phenylethyl alcohol was investigated by administering undiluted phenethyl alcohol to rats dermally at 0.07, 0.14, 0.28, 0.43 or 0.70 mL/kg/day from gestation day 6 through 15 at 24 hour intervals under an occluded dressing. Signs of irritation were noted in all dams dosed with the test material, dose-dependently for onset, severity and duration. No maternal deaths occurred. Clinical signs of toxicity (ptosis and/or urine stained abdominal fur) were noted in the dams dosed with 0.70 mL.kg bw/day of the test material. Live foetal body weights were statistically significantly lower in litters from dams dosed with the test material, but these were not biologically significant and likely a function of the increased numbers of fetuses per litter in treated groups. Significant increase in the incidence of foetuses with cervical ribs occurred for the 0.70 mL/kg/day group. These are reversible as are perturbations/delays in ossification caused by increased competition for nutrients as a result of increased foetal numbers in treated groups. Often the delays or perturbations in ossification were not treatment related. Under the conditions of the study, on the basis of no discernible adverse effects on fetal development, and ptosis and/or urine-stained abdominal fur in dams (and excluding irritation), the developmental and maternal no-effect levels for the test material were 0.7 and ≤0.43 mL/kg/day respectively. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).


 


Phenylethyl alcohol has been used as a read across substance since it is structurally similar to the substance to be registered. However it is expected to be more toxicologically active, and as such the results presented are taken to be the worst case scenario.


 


Justification for selection of Effect on developmental toxicity: via dermal route:


No single study selected since both studies are necessary to address effects on development through a weight of evidence approach.

Justification for classification or non-classification

According to the criteria outlined in Regulation (EC) No. 1272/2008 and Directive 67/548/EEC, the substance does not meet the criteria for classification for toxicity to reproduction. This is because high doses of the readacross-substance were required to elicit fetal effects, and maternal toxicity of the read-across substance is elicited at doses lower than those required to elicit developmental toxicity (a broad spectrum of non-specific malformations). The developmental toxicity is not a result of the maternal toxicity but rather is likely to stem from the same mechanisms that cause maternal toxicity (related to physical chemical / narcotic properties). Because it is considered that the substance for registration does not provoke these toxicities in adult animals up to the limit dose in 90 day repeat dermal dose toxicity studies, the developmental toxicity seen with the readacross substance will not manifest for the registered substance, and classification for reproductive effects is not waranted.

Additional information