2-hydroxy-1-(4-(4-(2-hydroxy-2-methylpropionyl)benzyl)phenyl)-2-methylpropan-1-one

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 July 2011 - 15 May 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

22 March 1996

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test

Version / remarks:

July 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

444-860-9

EC Name:

-

Cas Number:

474510-57-1

Molecular formula:

C21 H24 O4

IUPAC Name:

2-hydroxy-1-(4-{[4-(2-hydroxy-2-methylpropanoyl)phenyl]methyl}phenyl)-2-methylpropan-1-one

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Breeder: Harlan Netherlands
- Age (at start of treatment): 11 weeks
- Acclimatization: 7 days
- Body weight range: 291-371 g (males), 182-213 g (females)
- Diet: ad libitum
- Water: ad libitum
- Fasting: 16h before administration

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Carboxymethylcellulose sodium salt (0.5%)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
-The dose formulations were prepared weekly
- Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): As used in previous dose range-finding study

Details on mating procedure:

During the 14-day pairing period, females were housed with sexually mature males (1:1) until evidence of copulation was observed. The day with positive mating was designated day 0 post coitum.
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation was observed
- Proof of pregnancy: the daily vaginal smear was sperm positive, or a copulation plug was observed, referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were analyzed following an analytical procedure provided by the Sponsor and
adapted at Harlan Laboratories. The test item was used as the analytical standard. Analyzed
samples were not discarded without written consent from the study director.
Duplicates were taken of all samples and were stored at Harlan Laboratories Ltd., Füllinsdorf /
Switzerland. The samples were not discarded without written consent from the study director.

Duration of treatment / exposure:

Males: 4 weeks
Females: approx. 7 weeks

Frequency of treatment:

once daily

Details on study schedule:

First day of dosing: day 1 of pre-pairing
Pre-pairing: 14 days
Pairing: 14 days maximum
Gestation: approx. 21 days
End of treatment: day 4 post partum

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Doses / concentrations 4

Dose / conc.:

90 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Doses were selected based upon a previously conducted dose range-finding study in rats

Positive control:

not applicable

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy from 5 males from each group, blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes, 18h
- How many animals: 5 per sex and group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of the scheduled necropsy from 5 males from each group, blood samples from 5 lactating females from each group were obtained on day 5 post partum
- Animals fasted: Yes, 18h
- How many animals: 5 per sex and group
- Parameters checked in table [2] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: one time during the study
- Dose groups that were examined: 5 animals per sex and group
- Battery of functions tested: cage side observations, hand-held observations, open field observations, reflexes, hind / fore limb strength

Oestrous cyclicity (parental animals):

not determined

Sperm parameters (parental animals):

Spermatogenesis stages investigation and histopathology of interstitial cell structure.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no (not necessary)

PARAMETERS EXAMINED
The following parameters were examined in F1offspring: number and sex of pups, stillbirths, live births, presence of gross anomalies, survival rate, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
Dead pups, except those excessively cannibalized, were examined macroscopically.

Postmortem examinations (parental animals):

SACRIFICE
All animals sacrificed or found dead were subjected to a detailed macroscopic examination to establish, if possible, the cause of death. Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table)

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- All animals were subjected to postmortem examinations (macroscopic examination)

GROSS NECROPSY
- Gross necropsy consisted of a detailed macroscopic examination

Statistics:

Means and standard deviations of various data were calculated.
The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
Fisher's exact-test was applied if the variables could be dichotomized without loss of information.

Reproductive indices:

fertility indices, mean precoital time, post-implantation losses, mean litter size

Offspring viability indices:

pup sex ratios and viability indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

DAILY
Test item-related clinical signs were observed of males and females receiving 90 mg/kg body weight/day of Irgacure 127. Hunched posture, ruffled fur and discoloured feces were seen.

At the dose level of 90 mg/kg body weight/day, the male animal killed in extremies (no. 35) showed clinical signs such as ruffled fur from day 6 of treatment and hunched posture, ruffled fur and yellowish discoloured feces from day 8 of treatment. At necropsy no macroscopical finding was noted for this animal. Female no. 78, treated at the dose level of 90 mg/kg body weight/day and found dead on day 2 of the pairing period, had ruffled fur from day 6 of treatment and yellowish discoloured feces from day 9 of treatment. At necropsy examination, dark red iscoloration of the thymus, enlarged stomach and redish brown firm nodule in the jejunum were found. After histopathological examination it was concluded that the cause of death was likely the thrombosis of jejunum therefore this death was considered to be incidental.

At the dose level of 90 mg/kg body weight/day additional males were noted with ruffled fur and yellowish discoloured feces (no. 34 and 40) in the pre-pairing and pairing period. Male no. 37 had scabs and reddish sore on the right shoulder appearing in the pairing period and the scabs were noted in the after pairing period too.
Three additional females in this group (nos. 72, 73, 74) had ruffled fur starting in the pre-treatment period through pairing period. Female no. 73 had yellowish discoloured feces from the second week of pre-pairing period up to and including the gestation period. Female no. 72 also had huched posture on the first two days of the gestation period.
In groups 3 and 2 no clinical signs were noted in any sex during the study.

In group 1, male no. 10 had reddish sore at the cervical region for a short period in the pairing
period.


WEEKLY
At the dose level of 90 mg/kg body weight/day, the ruffled fur noted during the detailed weekly
clinical observations of males and females indicated a test item-related effect.

No test item-related effect was found in the other dose groups.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 90 mg/kg body weight/day, one male was killed for ethical reason on day 9 of treatment in the pre-pairing period and one female died spontaneously on day 2 of pairing period (on day 16 of treatment). After histopathological examination it was concluded that the cause of death of the female was likely the thrombosis in jejunum and, therefore, this death was considered to be incidental and not test item related. Histopathological examination of the male showed slight diffuse hyperkeratosis in forestomach and minimal erosion in glandular stomach, signs of moderate stress in thymus and adrenal glands as well as slight diffuse atrophy in the prostate, coagulating gland and seminal vesicles. The body weight and food consumption of this male was rapidly decreasing from the second day of treatment onwards probably due to individual sensitivity. Since other males at 90 mg/kg body weight/day, started to show body weight and food consumption reduction later in the treatment period (the body weight started to decrease from the pairing period onwards and the food consumption was decreased in the second week of pre-pairing period), relationship to the treatment could not be excluded.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

BODY WEIGHT MALES
Pre-pairing and Pairing Periods
From day 5 of the pre-pairing period the mean body weight of males receiving 90 mg/kg body weight/day of Irgacure 127 was minimally decreased throughout the study when compared to the control group, although not reaching a level of statistical significance.

The mean body weight gain of males receiving 90 mg/kg body weight/day of Irgacure 127 was lower from day 5 of pre-pairing period when compared to controls and it was statistically significantly reduced between days 8 and 14. In correlation with the low food consumption this was considered to be a test item-related effect. Thereafter mean values similar to the control group were achieved during the pairing and after pairing periods.

In groups 30 and 10 mg/kg body weight/day, mean body weights and mean body weight gains of the males were similar to that of the control group during the study.

BODY WEIGHT FEMALES
Pre-pairing, Pairing, Gestation and Lactation Periods
During the pre-pairing period the mean body weights of females at the dose level of 90 mg/kg body weight/day were minimally below of the control level achiving levels of occasional statistical significance from day 9 onwards. During the pairing, gestation and lactation periods mean body weights comparable to those of the control group were noted.

Significantly lower mean body weight gain was recorded from day 4 of the pre-pairing period was considered to be a test item-related effects. During the gestation period, minimally increased mean body weight gain values were found while there was no difference in mean body weight gain noted during the lactation period.

In groups 30 and 10 mg/kg body weight/day, mean body weights and mean body weight gains of the females were similar to that of the control group during the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

FOOD CONSUMPTION MALES
The mean food consumption of males receiving 90 mg/kg body weight/day of Irgacure 127 was statistically significantly lower between days 8 and 14 in the pre-pairing period and it was lower during the whole pre-pairing period. This was considered to be a test item-related effect.

The mean food consumption of males was at control level for each test item-treated groups after the pairing period.

In groups 30 and 10 mg/kg body weight/day, food consumption of males was not affected by the treatment of the test item.

FOOD CONSUMPTION FEMALES
The mean food consumption of females receiving 90 mg/kg body weight/day of Irgacure 127 was statistically significantly lower during the pre-pairing period which was considered to be a test item-related effect.

There was no effect of treatment with Irgacure 127 on the food consumption of females in the gestation or lactation periods.

In groups 30 and 10 mg/kg body weight/day, the mean food consumption of females was not affected by the treatment of the test item.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

MALES
No changes to be of toxicological relevance were noted in males in any dose group.

FEMALES
No changes were noted in females in any dose group.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The assessment of the biochemistry data did not reveal any test item-related effect in any dose
group in any sex.

MALES
No changes were noted in males in any dose group.

FEMALES
The total cholesterol level was statistically significantly higher in females treated at the dose level of 90 mg/kg body weight/day and lying slightly outside the range of historical control data, thus, test item-realted relationship cannot be excluded. The creatinine level was statistically significantly higher in females treated at the dose level of 10 mg/kg body weight/day however the value was within the historical background range. In absence of dose relationship no experimental relevance was attributed to this finding.

At the dose level of 30 mg/kg body weight/day no changes were noted.

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

FUNCTIONAL OBSERVATIONAL BATTERY
None of the parameters under investigation during the functional observational battery gave an indication of a test item-related effect.
Mean values of grip strength (fore- and hind paws) and landing foot splay gave no indication of
test item-related effects.
BODY TEMPERATURE
Mean body temperature in males was statistically significantly lower at the dose level of 90 mg/kg body weight/day compared to the control group (37.3 °C compared to 38.1 in the control group) and it was marginally lower when compared to historical control data range (37.5 - 39.1 °C). Additionally, slightly lower locomotor activity of this group was seen, thus a treatment-related effect can not be excluded.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Under the conditions of this experiment, the following treatment-related local irritative effects were induced:

Forestomach: hyperkeratosis in some females receiving 30 mg/kg body weight/day and all males receiving 90 mg/kg body weight/day and a single female at 90 mg/kg body weight/day, squamous cell hyperplasia with submucosal inflammation in a female at 30 mg/kg body weight/day; glandular stomach: focal erosion in one male at 10 mg/kg body weight/day and one male and female each at 90 mg/kg body weight/day, in the female associated with submucosal inflammation, mucosal atrophy.

Under the conditions of this experiment, the following treatment-related adaptive findings that are commonly seen in rats exposed with xenobiotics were induced:

Minimal to slight diffuse hepatocellular hypertrophy, characterized by an increase in
endoplasmic reticulum, was recorded in liver in all males and females at 90 mg/kg body
weight/day. This finding was considered to be adaptive.

Diffuse follicular cell hypertrophy in thyroid gland was noted in some males and females at 90 mg/kg body weight/day. This finding was considered to be consequent to the hepatocellular hypertrophy.

Histopathological examination of female no. 78 which died spontaneously receiving 90 mg/kg
body weight/day revealed that the cause of death was likely the thrombosis of jejunum and,
therefore, this death was considered to be incidental and not test item related.

Histopathological examination of the male no. 35 in the 90 mg/kg body weight/day group, which was killed in extremis, showed slight diffuse hyperkeratosis in forestomach and minimal erosion in glandular stomach, signs of moderate stress in thymus and adrenal glands as well as slight diffuse atrophy in the prostate, coagulating gland and seminal vesicles. The cause of moribundity could not be determind from the organs and tissues determined.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

LOCOMOTOR ACTIVITY
For females at the dose level of 90 mg/kg body weight/day, total level of locomotor activity was statistically significantly reduced. In males the total level of activity was lower when compared to controls or other test item-treated groups but it did not reach statistical significance. The reduced locomotor activity in animals receiving 90 mg/kg body weight/day of Irgacure 127 was considered to be a test item-related effect.
In females at the dose level of 30 mg/kg body weight/day the low beam count at 30 minutes was
lower when compared to control but the total level of activity was not affected in this dose group.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

MATING PERFORMANCE AND FERTILITY
Mating performance and fertility were unaffected by treatment.
All females mated within the first pairing period.
The pre-coital interval was unaffected by treatment with the test item.

CORPORA LUTEA COUNT
Mean number of corpora lutea per dam (determined at necropsy) was similar in all groups and
was unaffected by treatment.

GESTATION LENGTH
The mean gestation length was unaffected by treatment.

IMPLANTATION RATE AND POST IMPLANTATION LOSS
The number of implantation and the post-implantation loss were not affected by the treatment
with the test item.
The mean number of implantations of pregnant females was similar in all groups.
The mean incidence of post-implantation loss as a percentage of total implantations was 7.8, 7.9,
7.3 and 9.0 %, in order of ascending dose level.

Details on results (P0)

This study is a valid investigation of the toxicological effects resulting from repeated oral-gavage
administration of the test item Irgacure 127 to rats over at least 28 days. Irgacure 127 was
administered in 0.5% carboxymethylcellulose as vehicle at dosages of 10, 30, and 90 mg/kg
body weight/day, and controls received the vehicle only. Irgacure 127 was administered to male
rats for 28 days and to female rats for 14 days prior to pairing. Administration of the test item
continued through the pairing and gestation periods until the F1 generation reached day 4 post
partum.



At 90 mg/kg body weight/day, treatment with the test item resulted in a general reduction in food
consumption, body weight and body weight gain in males and females. Test item-related clinical
signs were observed in males and females such as hunched posture, ruffled fur and discoloured
feces. The locomotor activity was reduced in both sexes.
The postnatal loss was increased therefore the viability index was decreased. The mean number
of live fetuses on day 4 post partum was lower and the pup body weight development was
decreased. An increase of the liver weights of the dams (absolute and ratios) and in males was
observed and this correlated with diffuse hepatocellular hypertrophy which was associated with
consequent follicular cell hypertrophy of thyroid glands in some males and females at 90 mg/kg
body weight/day. The absolute and relative thymus weights of females were decreased and
correlated with macroscopical and histopathological changes such as thymic atrophy.
Degenerative stomach findings were present in some males and females at histopathological
examination.



At 30 mg/kg body weight/day, the mucosa in the stomach showed at necropsy to be thickened
with irregular surface in one female. The histopathology data showed correlating degenerative
lesions in the stomach. At 10 mg/kg body weight/day, a local irritative effect was noted in one
male. At 30 and 10 mg/kg body weight/day, no general or reproductive toxicity effects were
noted.



Based on the observed mortality, clinical symptoms, decreased body weight, decreased food
consumption, increased liver and decreased thymus weights at the dose level of 90 mg/kg body
weight/day the NOAEL (No Observed Adverse Effect Level) for general toxicity in parental
males and females was considered to be 30 mg/kg body weight/day.



Based on the significantly lower body weight of pups and the decreased viability index on day 4
post partum at the high dose level of 90 mg/kg body weight/day, the NOAEL for reproduction/
developmental toxicity was considered to be 30 mg/kg body weight/day.



Fertility index, conception rate, mean precoital time, number of implantations were not affected
by the treatment in any test item-treated group. No developmental effects were seen at dose
levels of 30 and 10 mg/kg body weight/day.



A NOAEL (No Observed Adverse Effect Level) for the local irritative effect of Irgacure 127 in
stomach could not be established for males and was established at 10 mg/kg bw/day for females
based on the microscopic findings detected.

Effect levels (P0)

1

Key result

Dose descriptor:

NOAEL

Remarks:

parental toxicity

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

organ weights and organ / body weight ratios

2

Key result

Dose descriptor:

NOAEL

Remarks:

fertility

Effect level:

90 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Target system / organ toxicity (P0)

Critical effects observed:

yes

Lowest effective dose / conc.:

30 mg/kg bw/day (nominal)

Organ:

liver

thymus

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Mean postnatal loss between days 0 and 4 post partum was higher (7.7 compared to 0.0 in
control or to the range of historical control data 0.0 - 3.8) and the mean number of living pup on
day 4 post partum was lower (10.5 compared to 11.9 in control, however it was within the range
of historical control data 10.3 - 13.2) in the group receiving 90 mg/kg body weight/day. Also the
viability index was statistically significantly lower in the high dose group (92.3 compared to
100.0 in the control or the range of the historical control data 96.2 - 100.0) therefore these were
considered to be test item-related effects.
At 30 and 10 mg/kg body weight/day, the postnatal loss and viability index were not affected by
the treatment of the test item.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean pup weights on day 1 post partum were unaffected by treatment with the test item. On day 1 post partum mean pup weights were 6.3, 5.7, 5.9 and 5.8 g in order of ascending dose level.

During the lactation period (on day 4 post partum), mean pup weight development resulted to be generally lower at the dose level of 90 mg/kg body weight/day (except for litter 71 resulting 12.7 g mean pup weight which was the highest in this study). Mean pup weights on day 4 post partum were 9.6, 8.4, 8.5 and 8.1 g in order of ascending dose level when included all relevant dams in evaluation. When excluding litters weighing more than 11 g mean body weight per group the mean body weight of pups were statistically significantly lower at the dose level of 90 mg/kg body weight/day. The mean values were as follows: 9.3, 8.4, 8.3 and 7.4 g in order of ascending dose level.
Therefore, the mean body weight of pups on day 4 post partum at the dose level of 90 mg/kg body weight/day was considered to be adverse test item related effect.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

No milk in stomach was found in one female pup in litter no. 67 (parent animals treated with the
dose level of 30 mg/kg body weight/day) on day 4 post partum. At the dose level of 90 mg/kg body weight/day, three male pups in three different litters (nos. 72, 74, 79) had no milk in stomach at first litter check and they were missing afterwards on day 4, 2, 2 post partum, 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

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings were noted at macroscopic examination of F1 pups.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

LITTER SIZE
The litter size was unaffected by treatment in any dose group.
The mean litter size of dams receiving 90 mg/kg body weight/day of Irgacure 127 was slightly lower when compared to controls (11.4 compared to 11.9) but was within the historical control data range (10.3 - 13.2).
SEX RATIOS
Sex ratios at first litter check and on day 4 post partum were unaffected by treatment with the test item.
The proportion of males on day 4 post partum was 48, 46, 53 and 49%, in order of ascending dose level.
For full breeding data see attached background material

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not examined

Details on results (F1)

Findings at First Litter Check and during Lactation
The mean litter size of dams receiving 90 mg/kg body weight/day of the test item was slightly lower when compaired to controls. Mean postnatal loss between days 0 and 4 post partum was higher and the mean number of living pup on day 4 post partum was lower in the group receiving
90 mg/kg body weight/day. However, only 2 litters out of 4 had a postnatal pup loss of higher than one (affected litters: 72, 74, 79, 80). The sex ratio was not affected. No abnormal pup was noted at any dose level.
At the dose level of 90 mg/kg body weight/day, three male pups in three different litters (nos. 72, 74, 79) had no milk in stomach at first litter check and they were missing afterwards on days 4, 2, 2 post partum, respectively.

Pup Weights Up to and Including Day 4 Post Partum
Mean pup weights on day 1 post partum were unaffected, whereas, the mean body weight of pups on day 4 post partum (dose level of 90 mg/kg body weight/day) was significantly lower and therefore considered to be a test item-related effect.

Macroscopical Findings
At necropsy of pups, there were no abnormal findings noted.

Effect levels (F1)

Dose descriptor:

NOAEL

Remarks:

developmental toxicity

Generation:

F1

Effect level:

30 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Group (mg/kg/day)	1 (0)	2 (10)	3 (30)	4 (90)
Female numbers	41-50	51-60	61-70	71-80
Number of females paired	10	10	10	10
Number of females mated (A)	10	10	10	9
Number of non-pregnant females (B)	2	1	0	1
Number of females, which died  before the scheduled necropsy (C)	0	0	0	1
Number of females, which did not deliver any pups (D)	0	1	0	0
Number of females which reared their pups until day 4 post partum	8	8	10	8

 

(A) Female no. 78 died spontaneously on day 2 in the pairing period
(B) Female nos. 45, 46, 51 and 77 were not pregnant
(C) Female no. 78 died spontaneously on day 2 in the pairing period
(D) Female no. 59 had 3 late embryonic losses only

Applicant's summary and conclusion

Conclusions:

Based on the observed mortality, clinical symptoms, decreased body weight, decreased food consumption, increased liver and decreased thymus weights at the dose level of 90 mg/kg body weight/day the NOAEL (No Observed Adverse Effect Level) for general toxicity in parental males and females was considered to be 30 mg/kg body weight/day.

Based on the significantly lower body weight of pups and the decreased viability index on day 4 post partum at the high dose level of 90 mg/kg body weight/day, the NOAEL for reproduction/developmental toxicity was considered to be 30 mg/kg body weight/day.

Fertility index, conception rate, mean precoital time, number of implantations were not affected by the treatment in any test item-treated group. No developmental effects were seen at dose levels of 30 and 10 mg/kg body weight/day.

A NOAEL (No Observed Adverse Effect Level) for the local irritative effect of the test item in
stomach could not be established for males and was established at 10 mg/kg bw/day for females based on the microscopic findings detected.

Executive summary:

The purpose of this guideline (OECD 422) study conducted with GLP certification, of the test material (EC: 444 -860 -9) was to generate preliminary information concerning the effects of the test item on the possible health hazards likely to arise from repeated exposure over a relatively limited period of time. In addition it provides information on possible effects on male and female reproductive performance such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

The test item was administered orally by gavage to male rats for 28 days and to female rats for 14 days prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum.

The test was conducted at three dose levels (10, 30, & 90 mg/kg bw/day). The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. Treatment related toxicity was observed - the pups were shown to have a lower bodyweight and a decreased viability index. The NOAEL was determined to be 30 mg/kg bw/day.

A standard dose volume of 10 mL/kg body weight with a daily adjustment to the actual body weight was used. Control animals were dosed with the vehicle alone (Carboxymethylcellulose sodium salt 0.5%).