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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
12 May - 23 June 2003
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance fatty acids, C18-unsatd., dimers (CAS No. 61788-89-4). In accordance to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004
Reference Type:
secondary source
Title:
Clubb, S. 2003. Dimer (CAS No. 61788-89-4) Reproduction/Developmental Toxicity Screening Test. Report Number 22828. Inveresk Research, Tranent, Scotland, as cited on the US EPA CHEMRTK website.
Author:
HPVIS - US EPA
Year:
2005
Bibliographic source:
http://www.epa.gov/chemrtk/pubs/summaries/ftadmrtr/c13651tc.htm

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimer (complex mixture of distillation products)
- Analytical purity: no data
- Physical state: light amber liquid
- Lot/batch No.: #PC-00-96
- Expiration date of the lot/batch: 19 June 2006
- Storage condition of test material: at ambient room temperature under nitrogen in the dark

Test animals

Species:
rat
Strain:
other: Sprague-Dawley Rat/IGS (Crl: CD®(SD) IGS BR)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd., Margate, Kent, UK
- Age at study initiation: ca. 8 weeks
- Weight on arrival: (P) Males: 145 - 165 g; (P) Females: 102 - 138 g
- Housing: Initially animals were housed 2 per polypropylene cage (42 x 72 x 20 cm) with stainless steel grid bottoms and mesh tops. A few days prior to pairing, males were transferred to individual grid-bottomed cages (58 x 38.5 x 20 cm). Mated females were housed individually in solid-bottomed cages (42 x 72 x 20 cm).
- Diet (e.g. ad libitum): Rat and Mouse Breeder Diet No. 3 Expanded Ground SQC (Special Diets Services Ltd., Stepfield, Witham, Essex, UK), ad libitum
- Water (e.g. ad libitum): domestic quality mains water, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 2
- Humidity (%): 50 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 12 May 2003 To: 23 June 2003

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: not applicable
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): Batches of diet were prepared at convenient intervals (weekly or fortnightly) during the study, and used within 12 days of preparation.
- Mixing appropriate amounts with (Type of food): An appropriate quantity of the test item was dissolved in a suitable volume of acetone. This solution was added to a suitable quantity of untreated diet and mixed for ca. 1 h with fan assisted venting to aid removal of the acetone. This dose premix was diluted with untreated diet to give the diet for the intermediate and high dose groups. The low dose diet was prepared by dilution of the intermediate dose diet with untreated diet. The diet premixes were then placed on a Winkworth mixer for ca. 20 min.
A control premix was prepared in the same way as the dose premix by using acetone and untreated diet. By dilution of this control premix with untreated diet the control diet was made, containing the same proportion of premix as the high dose diet.

Details on mating procedure:
- M/F ratio per cage: 1/1
- Length of cohabitation: overnight or up to 16 days
- Proof of pregnancy: copulatory plug and/or sperm in the lavage referred to as day 0 of pregnancy
- After 9 days of unsuccessful pairing replacement of first male by another male with proven fertility
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged (how): individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis of formulated diets were undertaken on 2 occasions during study treatment with regard to concentration and homogeneity. On each occasion triplicate samples of 50 g were withdrawn from each formulated diet (low, mid and high dose diet and control diet). The samples were analysed by the Toxicology Support Laboratory using a method supplied by the sponsor (no further data on method). ± 15% of the nominal concentration indicated an acceptable accuracy of formulation.

Results (mean found) of first analysis (in parenthesis: % difference from nominal):
200 ppm: 176 ppm (- 12%)
2000 ppm: 1770 ppm (- 11.5%)
20000 ppm: 16849 ppm (- 15.8%); reanalysis: 20862 ppm (+ 4.3%)

Results (mean found) of second analysis (in parenthesis: % difference from nominal):
200 ppm: 155 ppm (- 22.5%); reanalysis: 173 ppm (- 13.5%)
2000 ppm: 2109 ppm (+ 5.5%)
20000 ppm: 20609 ppm (+ 3.0%)

The low coefficient of variation (5.6% or less) was indicative of satisfactory homogeneity in all dose groups.
Duration of treatment / exposure:
(P) Males: at least 4 weeks overall, starting from 2 weeks prior to mating until termination
(P) Females: 2 weeks prior to mating, then through mating until at least day 4 of lactation
Frequency of treatment:
continuously
Details on study schedule:
no mating of F1 generation (not foreseen according to OECD 421)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 200, 2000, 20000 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
14.5, 147, 1450 and 16.5, 166, 1692 mg/kg bw/day for males and females, respectively
Basis:
other: group mean achieved dosages of test item calculated on food consumption, nominal dietary concentration and body weight (worst-case assumption of doses)
No. of animals per sex per dose:
10 P males and 10 P females per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
Existing relevant toxicological data was evaluated for dose selection. Dose levels take into account the maximum tolerable dose in the test animals and other factors such as anticipated exposure.

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: damaged teeth, piloerection, staining on dorsal neck/head, abnormal vocalisation, unkempt coat, body held low, hairloss, scabbing on head/mouth, agitation, hunched posture, rolling gait, pale discoloured skin on the whole body etc.

BODY WEIGHT: Yes
- Time schedule for examinations: All animals: one week prior to the start of treatment; Males: weekly throughout the treatment period until termination; Females: weekly until the start of mating period, then on day 0 of gestation, followed by day 7, 14 and 20 of gestation and day 1 and 4 of lactation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was determined as follows:
All animals: weekly until mating, starting one week prior to start of treatment; Males: weekly measurements continued over complete period, except during periods of co-habitation with females; Mated females: over the periods days 0-7, 7-14 and 14-20 of gestation and days 0-4 of lactation.
The achieved intake of the test item (mg/kg bw/day) was calculated from the following formula: achieved intake = (food consumption x dietary concentration, nominal) / body weight.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Once weekly throughout the study, the water consumption was monitored by visual inspection of the water bottles.

Oestrous cyclicity (parental animals):
Estrous cycle length was evaluated in P females by vaginal smears commencing on the day of pairing until mating had occured.
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of externally visible anomalies, body weight (day 1 and day 4 per sex and litter), physical or behavioural abnormalities, presence of milk in the stomach

GROSS EXAMINATION OF DEAD PUPS:
No for internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals (P): All surviving animals, when mating has been completed and the animals have been dosed for at least 4 weeks.
- Maternal animals (P): All surviving animals, when all observations have been completed, normally between day 4 and day 7 of lactation.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology was conducted on the epididymides, testes and ovaries of all control and high dose animals. Male reproductive organs (epididymides and testes) were weighed individually.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring were sacrificed between day 4 and day 7 of lactation.
- These animals were not subjected to postmortem examinations (macroscopic and/or microscopic examination).

Statistics:
Kruskal-Wallis non-parametric analysis (body weight, food consumption); analysis of variance and one-way analysis of covariance (organ weights);
Incidence data: Kruskal-Wallis analysis, Chi-squared test or Fisher's Exact Probability test;
Pairwise comparison (against control group): Fisher's F-protected LSD method, Student's t-test or Chi-squared protected z-test;
All statistical tests were two-sided and performed at the 5% significance level.
Reproductive indices:
fertility index (male) = number siring a litter / number paired;
fertility index (female) = number pregnant / number paired;
gestation index = number bearing live pups / number pregnant;
Offspring viability indices:
birth index = total number of pups born (live and dead) / number of implantation scars;
live birth index = number of pups live on day 0 of lactation / total number born (live and dead);
viability index = number of pups live on day 4 of lactation / number live on day 0;

Results and discussion

Results: P0 (first parental animals)

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
In the 2000 ppm dose group two female rats died prematurely. One animal was found dead after displaying several clinical signs, which included staining, piloerection, rolling gait, subdued behaviour, pale discoloured skin and a swollen/damaged tail. The other animal was killed prematurely due to a prolapse of the vagina. This female rat also showed hunched posture, pale eyes, subdued bahaviour and pale discoloured skin on the whole body. These two deaths were considered to be incidental.
Piloerection was noted in 1/10, 5/10, 3/10 and 8/10 males and in 3/10, 2/10, 1/10 and 2/10 females at dose levels of 0, 200, 2000 and 20000 ppm. Given the distribution of these findings and the lack of any dose relationship, it is not possible to associate them with the treatment.
The clinical observations for the other animals were considered to be consistent with those normally seen in rats of this age and strain.


BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
A statistically non-significant decrease in body weight was noted in males of the high dose group (313 ± 18 g) during the first week of treatment compared to the control males (331 ± 22 g). Weight gain thereafter was essentially similar to the control group (table 1). Among females of the high dose group, mean body weight gain was comparable to the controls throughout the study.
At 2000 ppm, there was a slight reduction in body weight gain in males. This minor difference in body weight performance was considered to reflect the low body weight noted at the start of treatment. No obvious effects of treatment in males at 200 ppm or in females at 2000 ppm and 20000 ppm were observed.


FOOD CONSUMPTION (PARENTAL ANIMALS)
In females treated with 20000 ppm mean food consumption was slightly increased during days 0-4 of lactation (29.2 g) compared to control (26.5 g). However, this increase was not significant. In males treated at this level, mean food consumption was essentially similar to control animals (table 1). At 2000 ppm there was a statistically significant increase in food consumption in female rats during the first week of treatment (controls: 18.2 ± 2.0 g; 2000 ppm: 20.2 ± 0.6 g). Thereafter food consumption was comparable to controls throughout the study (table 1). Since the increase in food consumption in female rats at 2000 ppm was not dose-dependent, this finding was not considered to be attributable to the administration of the test substance.
Mean food consumption in the low dose group was similar to that of control animals.


REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The male and female fertility index was 100% among all dose group. The median number of nights to a positive mating sign was, 2, 2, 4, and 3 nights for 0, 200 ppm, 2000 ppm, 20000 ppm, respectively. There were no animals passing one estrus to positive mating. The mean duration of gestation did not differ between control and treatment groups. Thus, there were no effects of treatment on mating performance, fertility or the duration of gestation.


ORGAN WEIGHTS (PARENTAL ANIMALS)
The testes and epididymides weights were essentially similar in all groups (table 1).


GROSS PATHOLOGY (PARENTAL ANIMALS)
In the female rat of the 2000 ppm dose group found dead all tissues were autolysed and the left horn of the uterus was enlarged. In another female of the same dose group killed prematurely due to a prolapse of the vagina a small thymus was examined.
Pelvic dilation in the right kidney in one female rat of the low dose group and distended urinary bladder in one male rat of the low dose group were considered not to be treatment-related. No further necropsy findings were noted in any of the dose groups.


HISTOPATHOLOGY (PARENTAL ANIMALS)
No abnormalities were noted by the examination of ovaries in the control and high dose females.
At the examination of testes, minimal seminiferous epithelial degeneration (bilateral) was observed in one male of the high dose group. No abnormalities in testes were seen in control rats. At the examination of the epididymis in the high dose group and control group chronic inflammatory cell infiltration was noted in three treated animals and in one control animal, respectively. In another control animal chronic inflammation (focal, adnexal) of the epididymis were seen. Minimal cellular debris (luminal, bilateral) was identified in one male at 20000 ppm. However, these findings in testes and epididymis were considered not to be treatment-related.


OTHER FINDINGS (PARENTAL ANIMALS): ACHIEVED DIETARY INTAKE
The achieved dietary intake for females in the high dose group was lower in the first week (1886 mg/kg bw/day) than in the second week (2190 mg/kg bw/day). Among males treated with 20000 ppm a higher mean dietary intake was achieved during the first week (1889 mg/kg bw/day) than in the following weeks (week 2: 1875 mg/kg bw/day; week 4: 1450 mg/kg bw/day).
In addition, there was a decreased concentration in females throughout the gestation period in all treatment groups (table 1). During this period, the achieved concentrations at all levels were slightly less than proportional to the diet concentrations.
At other times, the achieved concentration was essentially proportional to the diet concentrations.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Effect level:
>= 1 450 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: achieved intake equivalent to 20000 ppm (worst-case assumption); Basis: clinical signs; mortality; body weight; food consumption and compound intake; gross pathology; organ weights; histopathology; fertility index;
Dose descriptor:
NOAEL
Effect level:
>= 1 692 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOAEL
Effect level:
>= 1 692 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: achieved intake equivalent to 20000 ppm (worst-case assumption); birth index; live birth index; litter size; litter weight; pup weight; viability index; grossly visible abnormalities
Remarks on result:
other: Generation: developmental toxicity (migrated information)

Results: F1 generation

Details on results (F1)

VIABILITY (OFFSPRING)
The mean number of implants per pregnancy was higher in all the treated groups compared to the control (table 2). However, historical data shows that the findings in the treated groups were within background ranges for animals of this age and strain in similar studies performed at the testing laboratory. It was considered most likely, that the control value is at the lower end of the background range and thus, these findings were not treatment-related.
No obvious effects of treatment on litter size or litter survival in any of the dose groups were noted (table 2).

BODY WEIGHT (OFFSPRING)
There were no obvious effects of treatment on litter or pup weights in any of the dose groups (table 2).

OTHER FINDINGS (OFFSPRING)
There were no externally visible anomalies detected among all pups.

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 1: Reproductive/developmental toxicity screening test: parental examinations

 

Control group

200 ppm

2000 ppm

20000 ppm

Body weight gain (g)

Males

Week 0

288 ± 18

284 ± 14

277 ± 11

282 ± 15

Males

Week 1

331 ± 22

323 ± 12

320 ± 17

313 ± 18

Males

Week 2

370 ± 29

374 ± 18

358 ± 22

361 ± 24

Males

Week 3

404 ± 36

409 ± 19

387 ± 26

394 ± 29

Males

Week 4

432 ± 37

437 ± 23

411 ± 29

417 ± 39

Males

Week 0-4

144 ± 22

153 ± 13

134 ± 21

135 ± 25

Females

Week 0-2

41 ± 12

43 ± 8

38 ± 7

37 ± 7

Females

Day 0-20 of gestation

139

143

140

146

Females

Day 4 of lactation

287 ± 18

290 ± 12

294 ± 20

293 ± 22

Food consumption (g)

Females

Week 0

21.0 ± 1.2

18.0 ± 3.9

18.2 ± 2.0

18.2 ± 2.1

Females

Week 1

18.1 ± 2.8

17.9 ± 0.5

20.2 ± 0.6 **

19.1 ± 0.8

Females

Week 2

24.3 ± 3.2

21.5 ± 2.2

22.3 ± 1.9

24.2 ± 0.9

Females

Day 0-4 of lactation

26.5

22.7

27.9

29.2

Group mean achieved dosages of test item (mg/kg bw/day)

Males

Week 1

-

18.6

201

1889

Males

Week 2

-

18.4

177

1875

Males

Week 4

-

14.51

1471

14501

Females

Week 1

-

18.4

202

1886

Females

Week 2

-

19.9

204

2190

Females

Day 0-7 of gestation

-

19.4

189

1980

Females

Day 7-14 of gestation

-

18.6

184

1836

Females

Day 14-20 of gestation

-

16.51

1661

16921

Females

Day 1-4 of lactation

-

16.6

199

2097

Absolute epididymides weights (g)

Males

1.1258 ± 0.0809

1.1205 ± 0.0548

1.0607 ± 0.0611

1.0555 ± 0.1157

Absolute testes weights (g)

Males

3.39 ± 0.16

3.48 ± 0.32

3.36 ± 0.24

3.38 ± 0.35

** significantly different from control P<0.01

1 : These values represent the “worst-case” achieved intakes, which were taken into account for hazard assessment.

 Table 2: Reproductive/developmental toxicity screening test: Reproductive perormance, litter size and survival, litter and pup weights

 

Control group

200 ppm

2000 ppm

20000 ppm

Mean duration of gestation and overall litter performance values

Mean number pregnant

10

10

10

10

Mean duration of gestation (days)

21.7

21.8

21.6

21.7

Number of females producing a liver litter

10

10

9

10

Gestation index (%)

100

100

90

100

Mean number of implant sitesa per pregnancy

12.9± 2.3

15.6 ± 0.8

14.7 ± 2.6

15.2 ± 1.9

Mean total number of pupsa born per litter

11.8 ± 2.0

14.1 ± 1.6

13.0 ± 1.8

14.1 ± 1.6

Mean number of live pups per litter

Day 0 of lactation

11.8 ± 2.0

14.1 ± 1.6

12.9 ± 2.0

13.7 ± 1.8

Mean number of live pups per litter

Day 4 of lactation

11.6 ± 2.0

12.1 ± 3.4

12.6 ± 2.1

12.0 ± 3.9

Group mean F1 survival indices

Birth index (%)

91

91

91b

92

Live birth index (%)

99

100

99b

93

Viability Index Days 0-4(%)

79

87

86b

82

Group mean litter and pup weighta

Litter

Day 1 of lactation (g)

78 ± 10

73 ± 22

79 ± 12

82 ± 11

Litter

Day 4 of lactation (g)

112 ± 13

105 ± 30

116 ± 18

111 ± 35

Males

Day 1 of lactation (g)

7.0± 0.8

6.0 ± 0.9

6.5 ± 0.6

6.4 ± 0.9

Males

Day 4 of lactation (g)

10.0 ± 1.3

8.9 ± 1.1

9.5 ± 0.9

9.0 ± 1.1

Females

Day 1 of lactation (g)

6.5 ± 0.8

5.6± 0.9

6.2 ± 0.5

6.1 ± 1.0

Females

Day 4 of lactation (g)

9.5 ± 1.3

8.5 ± 1.2

9.1 ± 0.7

8.8 ± 1.3

a: excludes litters where all pups died

b: based on 8 litters (litters of animal found dead and of animal killed prematurely were not considered)

Applicant's summary and conclusion