Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23. May 2003 - 05. Aug. 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: (P) Males: 185.9 - 201.0 g; Females: 160.8 - 182.3 g
- Fasting period before study: no
- Housing: The animals were housed in a limited access rodent facility. During the pre-mating period, animals were housed up to 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent paper which was inspected and changed at least 3 times a week. During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42x26x18cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were be fore mating. After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18cm (Techniplast Gazzada S.a.r.l., Buguggiate, Varese). Suitable nesting material was provided and was changed at least 3 times a week.
- Diet: A commercially available laboratory rodent diet (Altromin MT pelleted diet, D-32770 Lage, Postfach 1120, Germany) was offered ad libitum throughout the study.
- Water: ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs
Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: prepared daily in polyethylene glycol (PEG 400)
VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle in toxicology studies
- Concentration in vehicle: 10, 30, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of mating until sperm identification and/or copulation plugs were found. The pairing combinations of any animals which have not had positive identification of mating after 14 days of pairing will be changed within each treatment group. The subsequent pairing will be monitored for mating as described above for a maximum period of 14 days. If no mating oceurs the females will be killed at least 7days after the last day of the mating session.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory . Sampies of the formulations prepared during the first and the last week of treatment were analysed to check the concentration and the homogeneity.
Duration of treatment / exposure:
Males: Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks prior to pairing and thereafter through the day be fore necropsy. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post-partum periods, until the day before necropsy (Day 4 post-partum). During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post-coitum and on Day 1 post-partum. Thereafter individual dose volumes remained constant.
Frequency of treatment:
once daily
Details on study schedule:
On the day of allocation, 7 days prior to the start of treatment, all animals were weighed. Animals at the extremes of the weight distribution were excluded to leave the required number of animals. The rats were allocated to the 4 groups by computerised stratified randomisation to give approximately equal initial group mean body weights. Males: Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks prior to pairing and thereafter through the day be fore necropsy. Females: Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during gestation and post-partum periods, until the day before necropsy (Day 4 post-partum).
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Details on study design:
- Dose selection rationale: Dose selection on basis of a confirmatory study (RTC 40330EXT)
- Rationale for animal assignment (if not random): random
Positive control:
none
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice a day on working days, and once on weekends and public holidays. Examination of individual animals for signs of reaction to treatment was carried out daily, prior to dosing, 30 minutes, 1 and 2 hours after dosing.
- Full records were maintained for all measurements and observations.


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once per treatment week, and whenever possible at 7 day intervals, each animal was observed and any clinical signs were recorded.


BODY WEIGHT: Yes
- Time schedule for examinations: Males: Animals were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females: Animals were weighed on the day of allocation to treatment groups, weekly from the first day of treatment to mating, on Days 0, 7, 14 and 20 post-coitum and on Days 1 and 4 postpartum.


OTHER:
Oestrous cyclicity (parental animals):
not examined
Sperm parameters (parental animals):
not examined
Litter observations:
As soon as possible after parturition (Day 0 or 1 post-partum), the total litter size (live and dead) was counted, live pups were individually identifIed within the litter, sexed and examined for external abnormalities. All litters were weighed on Day 1 post-partum. All litters were examined daily for dead and abnormal pups. The pups were also weighed on Day 4 post-partum. All pups found dead were necropsied.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: The males were killed after the mating of females with carbon dioxide.
- Maternal animals:The females with live pups were killed on Day 4 post-partum with carbon dioxide. Females which had not given birth 25 days after the positive identification of mating were killed shortly after.


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


HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues of abnormalities, seminal veshicles. epididymides, testes, prostate gland, ovaries, uterus and vagina were preserved. Detailed histopathological examination was performed on ovaries, testes, and epididymides from the control and the high dose groups.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at [#?] days of age.
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Necropsy of pups also comprised the confirmation of sex by gonadal inspection.


HISTOPATHOLOGY / ORGAN WEIGTHS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
SACRIFICE
- All viable pups were euthanised by intrascapular injection of Tanax on Day 4 post-partum.


GROSS NECROPSY
All pups found dead in the cage were necropsied. All live pups were killed on Day 4 postpartum and examined for the following:
a) external and internal abnormalities;
b) sex confirmation by gonadal inspection.
Statistics:
For continuous variables the significance of the differences amongst group means was assessed by Dunnett's test or a modified t test, depending on the homogeneity of data. The homogeneity of the data was assessed by Bartlett's test. Statistical analysis of non-continuous variables was carried out by means of the Kruskal-Wallis test and intergroup differences between the control and treated groups assessed by a non-parametric version of the Williams test. The mean values, standard deviations were calculated from actual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
Fate of females: Two unscheduled deaths occurred during the study: one female in group 2 (animal no. 15090033) was sacrificed for humane reasonson Day 9 post-coitum and another female of the same group (animal no. 15090021) was found dead on Day 0 post-partum. A total of 14 live pups from this latter dam were humanely sacrificed. A total of 10 females were found not pregnant at necropsy, 4 in the control group, 1 in group 2 which wassacrificed for humane reasons on Day 9 post-coitum, 2 in group 3 and 3 in group 4. One female in group 4 (animal no. 15090065) had total litter losson Day 0 postpartum. The number of females with live young at Day 4 post-partum was 6 to 8.
Clinical signs and pre- and post-dose observations: Clinical signs observed at weekly intervals in FO males and females were limited to common
conditions of the skin and fur. Rales were occasionaIly observed at pre- and post-dose observations, in controls and in groups 2 and 4 male animals, and in groups 3 and 4 females (data not tabulated). Soft faeces were recorded during cages observation for control and treated groups.
Body weights: A statistical significant lower body weight was observed in male animals of group 2 compared to controls on study day 15 (dosing phase). This difference was not considered treatment related since no other differences in body weight and/or body weight gain were noted between the treated and control groups. Body weight and body weight gain for female animals were generaIly comparable between the treated and control groups before mating and during gestation and post-partum periods.
Food consumption: Food consumption was unaffected by the treatment.
Reproductive parameters: Irregular oestrus cycles were observed in treated groups and also in controls. However, a high incidence of animals with irregular oestrus cycles was noted in the treated groups compared to controls. This finding was not considered to be of toxicological significance
since no effects on relevant fertility parameters were observed. The copulatory and fertility index as weIl as the pre-coital intervals were not affected by the treatment.
Implantation, pre-birth loss data and gestation length: The gestation periods were similar between the groups. No significant differences were observed for the number of implantations, corpora lutea and pre-birth loss in treated groups compared to controls.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: overall results; body weight, body weight gain, food consumption, fertility index, pre-coital interval, copulatory index, histopathology of ovaries and testes, litter development
Remarks on result:
other: Generation not specified (migrated information)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Litter data: The mean litter and pup weights were sligthly reduced in group 4 compared to controls at birth as weIl as on Day 4 post-partum.
Sex ratios: No statistically significant differences were observed in the sex ratios between the groups.
Pre-weaning clinical signs of Fl pups: Pre-weaning clinical signs did not show treatment-related effects.
Necropsy findings in decedent pups: Decedent pups were found in treated groups including controls. One female in group 2 was found dead on Day 0 post-partum and a total of 14 pups were humanely sacrificed. No abnormalities were noted at necropsy for these pups (data not presented). One female in group 4 had total litter loss. No relevant fmdings that could be treatment-related were reported.
Necropsy findings in Fl pups at Day 4 post-partum: No significant fmdings were observed between the groups at necropsy of the pups.
Absolute and relative organ weights in FO males: Statistically significant higher relative organ weights of epididymides and testes were observed in group 2 compared to controls. These differences were not considered to be of toxicological relevance since no dose-dependency was observed.
Macroscopic observations: No macroscopic observation was reported in the treated animals that could be considered to be treatment-related.
Microscopic observations: The histopathological examination of the ovaries, testes and epididymides did not reveal any evident differences between the findings observed in the treated and control animals that could be considered treatment-related. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No abnonnalities were noted.
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
> 1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
not specified

Litter data at birth and on Day 4 post-partum - Group mean data

 

 

At birth

On Day 4

Group

 

Litter total

Live litter size

Pup loss %

Litter wt (g)

Mean pup wt (g)

Live litter size

Cumulative loss %

Litter wt. (g)

Mean pup wt. (g)

1

(n)

6

6

6

6

6

6

6

6

6

 

Mean

14.7

14.2

3.5

86.6

6.4

13.3

9.3

128.6

9.6

 

SD

1.2

1.5

3.9

16.5

0.5

1.6

4.3

28.4

1.1

 

 

 

 

 

 

 

 

 

 

 

2

(n)

9

9

9

8

8

8

8

8

8

 

Mean

14.3

13.9

3.8

87.4

6.5

13.3

8.8

122.9

9.4

 

SD

2.7

3.1

7.9

18.9

0.5

3.3

8.4

26.2

1.2

 

 

 

 

 

 

 

 

 

 

 

3

(n)

8

8

8

8

8

8

8

8

8

 

Mean

14.0

13.4

4.0

82.3

6.4

13.0

6.3

120.0

9.5

 

SD

3.9

3.7

7.0

17.5

0.8

3.6

9.3

23.6

1.4

 

 

 

 

 

 

 

 

 

 

 

4

(n)

7

7

7

6

6

6

6

6

6

 

Mean

14.6

11.3

22.2

69.6

5.9

11.7

19.1

100.6

8.8

 

SD

1.8

5.7

35.6

12.4

0.6

2.7

12.0

12.8

1.2

 

 

 

 

 

 

 

 

 

 

 

* = Statistically significantly different from control group value at p < 0.05

Conclusions:
On the basis of the results, the dosage of 1000 mg/kg/day could be considered as the NOAEL.
Executive summary:

The effects on reproduction and development of the offspring were evaluated after oral administration of BENZOIC ACID ISONONYLESTER in male and female rats. The test item was given before and during mating and throughout the gestation period until Day 3 post-partum at dosages of 100, 300 and 1000 mglkg/day. Body weight, body weight gain and food consumption were not affected by treatment in male or female animals. Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls. A slight reduction in mean pup weight and consequently in litter weight was noted in group 4 females at birth and on Day 4 post-partum. The histopathological examination of the ovaries, testes (including the stage in the spermatogenic cycle) did not reveal any evident differences between the findings observed in the treated and control animals. On the basis of the results, the dosage of 1000 mg/kg/day could be considered as the NOAEL.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Species:
rat
Additional information

OECD 421 Screening Reproduction Development: In this study, the effects on reproduction and development of the offspring were evaluated after oral administration of BENZOIC ACID ISONONYLESTER in male and female rats. The test item was given before and during mating and throughout the gestation period until Day 3 post-partum at dosages of 100, 300 and 1000 mg/kg/day body weight, body weight gain and food consumption were not affected by treatment in male or female animals.

Fertility: Reproductive parameters such as fertility index, pre-coital interval and copulatory index did not show significant differences in treated groups compared to controls.

The test item benzoic acid isononylester did not induce any effects on fertility parameters like fertility index, pre-coital interval and copulatory index in an OECD 421 study up to the maximum dose of 1000 mg/kg/day.


Short description of key information:
OECD 421 screening study: NOAEL >= 1000 mg/kg/day

Effects on developmental toxicity

Description of key information

OECD 421 screening study: NOAEL >= 1000 mg/kg/day
OECD 414 with rats: NOAEL = 300 mg/kg/day
OECD 414 with rabbits: NOAEL = 150 mg/kg/day

OECD 443:

NOAEL (general toxicity parental males) = 1000 mg/kg/day

NOAEL (general toxicity parental females, reproductive and developmental toxicity of Cohort 1A, Cohort 1B and F2 males and females): 300 mg/kg/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
chronic
Species:
rat
Additional information

OECD 421 Screening Reproduction Development:

Developmental effects: in this study with rats, a slight reduction in mean pup weight and consequently in

litter weight was noted in group 4 females at birth and on Day 4 post-partum. The histopathological examination of the ovaries, testes (including the stage in the spermatogenic cycle) did not reveal any evident differences between the findings observed in the treated and control animals.

OECD 414 Prenatal Developmental Toxicity Study in rats: 3 groups of 24 mated female rats were used in the study to assess the effects

of BENZOIC ACID ISONONYLESTER on pregnancy and embryo-foetal development in the rat during gestation. The rats received BENZOIC ACID ISONONYLESTER at dosages of 100, 300 and 1000 mg/kg/day, respectively. A similarly constituted group (Group 1) received the vehicle alone (corn oil) and acted as a control. The animals received the test item or the vehicle from Day 0 to Day 19 post coitum. An increased incidence of scabs was noted in the high dose females when compared to the control group. Statistically significant decreases in body weight were observed in the high dose group on Days 18 and 20 post coitum. Changes in body weight gain were also noted in the high dose group. A statistically significant reduction in terminal body weight, uterus weight, absolute weight gain and litter and mean foetal weight was observed in the high dose group. A general retardation of the ossification of the skull, 5th and 6th sternebrae, thoracic centra, pubis bones and sacral arches was observed in the high dose group at skeletal examination. These changes were considered to be a consequence of the presence of the foetuses with a lower foetal weight and due to the signs of marginal maternal toxicity observed in the high dose group. On the basis of these results the mid- dose level (300 mg/kg/day) could be considered as the NOAEL for both maternal and foetal toxicity.

OECD 414 Prenatal Developmental Toxicity Study in rabbits: The maternal and embryo-foetal toxicity of the test item was investigated following daily oral administration to pregnant rabbits during the gestation period. The test item was administered to 3 groups of 20 mated female rabbits from Day 6 through Day 28 post coitum at dosages of 50, 150 and 450 mg/kg/day. A group of 20 mated female rabbits served as controls and received the vehicle during the treatment period (corn oil). All surviving females were caesarean-sectioned on Day 29 post coitum and subjected to a post mortem examination. The number of corpora lutea, implantations, early and late intrauterine deaths, live and dead foetuses, uterus weight and foetal weight were reported. All foetuses were examined externally and internally. In addition, examinations of fixed head and skeletal bones of foetuses were performed.

Mortality: A total of eight females were sacrificed for humane reasons: one control female, three low dose females and two mid- and high dose females. In addition, one control female and one mid-dose female were found dead on gestation Days 25 and 28, respectively.

Fate of females: Abortion occurred in two females, one of the low and one of the mid-dose group, both on gestation Day 28 and in two females of the high dose group on gestation Days 21 and 22. Premature birth on gestation Day 28 was noted in one control female, one low dose female and one mid-dose female. This mid- dose female was found dead after the premature birth. In addition, premature birth was also noted in another mid-dose female on gestation Day 25 and in one high dose female on gestation Day 29. Total resorption and unilateral implantation were detected in two different low dose females. One control female was found not pregnant at necropsy. The number of females with live foetuses on gestation Day 29 was 17 in each group.

Clinical signs and pre- and post-dose observations: Reduced food and water intake and reduced faeces were noted in the control and treated females. Red staining and foetuses were found on the cage tray of the females which gave birth prematurely. Dyspnoea and cold to touch were also noted in some of the females which were sacrificed for humane reasons. Breathing difficulties were observed in one female of the control group and one female of the low dose group, at post-dose observations, soon after dosing and at 1 hour after dosing. No other reactions to treatment were noted at the post-dose examinations performed.

Body weight and body weight gain: A slight, but statistically significant reduction in body weight was noted in mid-dose females from allocation until gestation Day 18, when compared to controls. This reduction was compensated within the following testing days and body weight was comparable to the control group by the end of the study. Body weight gain was not affected by treatment.

Food consumption: A decrease in food consumption was noted in the control and treated females during the treatment period.

Terminal body weight, uterus weight and absolute weight gain: A statistically significant reduction in gravid uterus weight was noted in the high dose group when compared to controls. No significant changes were seen in terminal body weight and absolute weight gain.

Litter data and sex ratios: A statistically significant increase in the intrauterine deaths at early stages of gestation and pre-implantation loss was noted in the high dose females when compared to controls. In addition, a slight but statistically significant reduction in the number of male foetuses was also noted in this group. This difference is considered as a consequence of a general decrease in the total number of live foetuses rather than as an effect on sex ratios. In all treated groups, a statistically significant reduction was detected in the intrauterine deaths at late stages of gestation, however this difference is due to the high incidence of this parameter in the control group.

Macroscopic examination: None of the findings, reported at necropsy of the unscheduled dead animals and of those sacrificed at termination, showed any relation with the treatment and were thus considered to be incidental or spontaneous in origin.

External and internal examination in foetuses: A total of 125 small foetuses were present at necropsy: 37 in the control group, 29 in the low dose group, 31 in the mid-dose group and 28 in the high dose group. In the low dose group one foetus showed some malformations such as umbilical haernia, dextrocardia and agenesis of kidney and ureter. In the high dose group, a total of 4 foetuses from three different litters showed malformations: one foetus (dam no. 123) showed extreme pelvic dilatation and forepaw flexure. In the litter of dam no. 155 one foetus showed hindpaw flexure and a second foetus presented hindlimb flexure. In the litter of dam no. 157 another foetus showed hindlimb flexure.

Fixed head examination in foetuses: No treatment-related changes were seen at fixed head examination between the control and treated groups.

Skeletal examinations in foetuses: No changes that could be considered treatment-related were noted at the skeletal examination of the foetuses between the control and treated groups.

On the basis of these results, the mid-dose level of 150 mg/kg/day of the test item BENZOIC ACID ISONONYLESTER could be considered as the No Observed Adverse Effect Level (NOAEL).

OECD 443 Extended One-generation Reproductive Toxicity Study (EOGRTS) in rats (Cohorts 1A and 1B with the extension to a 2nd generation):

The pre- and postnatal effects of BENZOIC ACID ISONONYLESTER on development, as well as a thorough evaluation of systemic toxicity in pregnant and lactating females and young

and adult offspring were investigated. In this study, the test item was oral administered in male and female rats (parental generation and Cohort 1A, 1B) with dosages of 100, 300 and 1000 mg/kg/day.

After weaning of F1, pup mortality occurred in the high dose group, which was the reason to reduce the high dose to 500 mg/kg. The following effects were observed:

Parental toxicity:

The study started with groups of 25 rats per sex. A total of two female unscheduled deaths were observed, but without any clear reason or dose-dependency. No treatment related clinical signs were observed in parental males with the exception of salivation noted in three high dose males (1000 mg/kg/day) in isolated occasions. There was a decrease in food consumption in high dose females during lactation. Increase of neutrophils and decrease of eosinophils were recorded in a number of animals of both sexes in the high dose. Moreover, a slight increase of neutrophils was recorded in males of the mid dose. Fluctuations of some liver and/or metabolic markers were recorded in a number of treated animals of both sexes in the high dose group. These findings matched with some liver alterations observed at the histopathological examination. Due to the absence of degenerative changes in the liver, these alterations were considered as adaptive and not adverse effects. A minimal reduction in the terminal bodyweight was noted in high dose males. Absolute and relative liver weights were increased, with statistical significance, in high dose males (approximately 22% or 25%) and females (about 15%), when compared with controls. A slight statistically significant increase in absolute and relative kidney weights was also recorded in high dose males. At post mortem examination, treated animals, particularly those receiving the dose levels ≥ 300 mg/kg/day, showed swollen and enlarged liver. Treatment-related microscopic findings were only seen in the liver of most males and in some females of the high dose group. The treatment related liver changes consisted of minimal to mild centrilobular hepatocytic hypertrophy, associated with cytoplasmic eosinophilia, morphologically represented by an enlarged size of the hepatocytes possibly correlated to the hepatic enzyme induction. In the absence of degenerative changes, such liver findings were considered adaptive and not an adverse change.

Developmental effects in the parental generation:

Fertility index in the mid and high dose animals was decreased, though without dose dependency. A decrease in number of implantation sites, total litter size at birth and live litter size at birth was noted in high dose females. In particular, the decrease between total litter size at birth versus the mean number implantations caused an increase both in pre-birth loss and pre-natal loss data.

Developmental effects in F1:

A decrease in total litter size and live litter size at birth were recorded in high dose females. During lactation, live litter size and litter weight were decreased in high dose females. Mean pup weight was also decreased between Days 1 and 21 p.p. reaching statistical significance decrease at the second half of lactation. The highest postnatal loss in the high dose group occurred between birth and day 4 p.p. During the lactation period, the total number of lost pups (found dead or missing pups) in the high dose group was increased. An increase in anogenital distance noted in female high dose pups treated at 1000 mg/kg/day was considered to be of slight severity and not clearly related to the treatment. Immediately after weaning and starting with direct dosing of the pups, unexpected pup mortality was observed in the high dose group (5 pups). Starting from nominal Day 28 and until nominal Day 63 (males) or Day 56 (females), animals receiving 500 mg/kg/day showed a lower body weight, when compared to the control group. In cohort 1A, a trend toward a delay in vaginal opening and the first oestrous was noted in all treated groups. Although no relation to the doses was recorded, this delay

reached statistical significance in the low and high dose groups. In cohort 1B, vaginal opening and first oestrous were statistically significantly delayed in females receiving 500 mg/kg/day. Male rats of both cohorts receiving 500 mg/kg/day showed a statistically significant delay for balano-preputial separation. However, body weight on the day of achievement of sexual maturation in both sexes was not influenced, indicating a general retardation in development as the reason for these observations. A minimal reduction in terminal body weight was noted only in F1 females dosed at 500 mg/kg/day (high dose). During the maturation phase, lower body weights were observed in males receiving 500 mg/kg/day and in females receiving the dose levels ≥ 300 mg/kg/day. This effect was more evident in females than in males on nominal Day 28 with a general trend of recovery thereafter until the end of the maturation phase. On nominal Day 28 of the mating phase, body weight gain of males receiving 500 mg/kg/day was statistically significantly decreased (-29%) compared to the control. Before pairing, during the maturation phase, females receiving the dose levels ≥ 300 mg/kg/day showed a statistically significant decrease in body weight gain on nominal Day 35.

Conclusions:

The second breeding of Cohort 1B animals was triggered by the adverse effects observed in parental animals and F1 progeny at the dose level of 1000 mg/kg/day.

Taken into consideration the results obtained from this study, the NOAEL (No Observed Adverse Effect Level) for general toxicity for Parental males was selected at 1000 mg/kg/day,since the treatment-related liver changes noted at microscopic observations were considered adaptative and not adverse.

The NOAEL for general toxicity for Parental females, F1 (Cohort 1A and Cohort 1B males and females) and for reproductive and developmental toxicity of F2 pups was considered to be 300 mg/kg/day based on the following effects recorded in Parental animals and F1offspring firstly dosed at 1000 mg/kg/day: No severe signs of maternal toxicity in parental females were noted. However, the

reduction in litter size observed in F0 generation could be considered as primary response to the treatment with the test item and not a secondary effect. Postnatal mortality of F1 pups was also associated with offspring decreased bodyweight at birth and until weaning. Severe clinical signs incl. death of 5 pups were also noted in selected F1 pups dosed at weaning firstly at 1000 mg/kg/day.

A delay in the age at which sexual maturation was attained both in male and female in F1pups (opening of the vagina, first oestrous, preputial separation) was also recorded, although in the absence of body weight change performed on the same day in which the maturation occurred, indicating that these effects are probably secondary to a general delay in development. It is important to notice that, after decreasing the high dose to 500 mg/kg, no parental or developmental effects were observed in F1 offspring (the lower body weight at weaning at 1000 mg/kg could not be completely recovered, however, body weight gain was unaffected afterwards).

Justification for classification or non-classification

GHS classification (GHS UN rev.7, 2017): classification as reproductive toxicants Cat. 2 required (H361d).

The classification is based on adverse developmental effects of F1 pups in the Extended One-Generation Reproductive Toxicity Study (EOGRTS) in rats:

The first developmental effects like decreased litter size were observed in the high dose group. The most striking effect, however, is the unexpected mortality in high dose pups after weaning and starting of direct administration to the pups. It is important to note that after reducing the high dose group to 500 mg/kg, no more effects were observed (the delay in development, also probably leading to the delayed sexual maturation in both sexes, is still a consequence of the 1000 mg/kg treatment which, despite of a normal body weight gain after reducing the dose, could not cause a complete recovery of body weights). The critical situation is: quite striking effects in the high dose developmental parameters in the absence of clear parental effects. Due the absence of any effects after reducing the high dose to 500 mg/kg/day, it is worthwhile to suggest R2(D) as a self-classification.