Acetic acid, 2,2-difluoro-2-[[2,2,4,5-tetrafluoro-5-(trifluoromethoxy)-1,3-dioxolan-4-yl]oxy]-, ammonium salt (1:1)

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

Sprague-Dawley rats, Reproduction/Developmental Toxicity Screening (OECD 421, GLP)

Dosage (oral gavage): 0, 5, 20, 60 mg/kg/day (males); 0, 5, 20, 80 mg/kg/day (females)

NOAEL fertility parameters: 60 mg/kg/day (male) (no effects on fertility parameters)

NOAEL fertility parameters: 80 mg/kg/day (female) (no effects on fertility parameters)

NOAEL reproduction parameters: 20 mg/kg/day (female) (maternal toxicity at 80 mg/kg/day, effects on pregnancy and early lactation)

NOAEL postnatal development: 20 mg/kg/day (litters lost at 80 mg/kg/day not allowing evaluation)

NOAEL maternal toxicity: 20 mg/kg/day (based on significantly lower body weight, body weight gain, food consumption during the gestation period at 80 mg/kg/day)

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-02-03 until 2011-04-12.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

27 July 1995

Deviations:

no

Remarks:

No deviation on the performance of the main study. An additional satellite group was treated for 28 days to assess organ toxicity.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 100 Hsd: Sprague Dawley SD rats (50 males and 50 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were obtained by Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy.
Body weight ranges were 182 to 201 g for males and 155 to 166 g for females. An acclimatisation period of 19 days was allowed before the start of treatment.

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C ± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 25 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
During the pre-mating period, animals were housed 5 of one sex to a cage, in clear polycarbonate cages measuring 59x38.5x20 cm with a stainless steel mesh lid and floor.
During the mating period, animals were housed on the basis of one male to one female in clear polycarbonate cages measuring 42x26x18 cm with a stainless steel mesh lid and floor. The males were re-caged after mating as they were before mating.
After mating, the females were transferred to individual clear polycarbonate solid bottomed cages measuring 42x26x18 cm. Suitable nesting material was provided and changed at least 3 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially rodent diet 4 RF 21 was offered ad libitum.

On the day of allocation (6 days prior to the start of treatment) all animals were weighed. Animals at the extremes of the weight distribution and some animals showing minor signs of ill health were excluded to leave the required number of animals. The rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights.
Individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed up to 5 of one sex per cage.
The cages were identified by a label recording the study number, animal numbers and details of treatment.
The arrangement of cages in batteries was such that cages from each treatment group was evenly distributed across the battery to minimise possible environmental effects.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified water

Details on exposure:

The test item was administered orally by gavage at a dose volume of 10 ml/kg body weight.
Control animals received the vehicle alone at the same dose volume.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until sperm identification and/or copulation plugs were found.
The pairing combination of the animal which did not have positive identification of mating after 14 days of pairing was changed within its treatment group. The subsequent pairing was monitored for mating as described above until indications of pregnancy were evident.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

Males (Main groups): Male animals of the main groups were dosed for 2 consecutive weeks prior to pairing and thereafter through to the day before necropsy, for a total of 6 consecutive weeks.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Females (Main groups): Female animals of the main groups were dosed for 2 consecutive weeks prior to pairing and thereafter during gestation and post partum periods up to Day 3 post partum (including the day of parturition), with the exception of Group 4 dams, which were sacrificed between Day 0 and Day 1 post partum, due to total litter loss.
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.
Males and Females (Satellite group): Animals of the satellite group were dosed for a minimum of 28 consecutive days.

Frequency of treatment:

once a day, 7 days a week

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Males / dry salt

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Males / dry salt

Dose / conc.:

60 mg/kg bw/day (nominal)

Remarks:

Males / dry salt

Dose / conc.:

5 mg/kg bw/day (nominal)

Remarks:

Females /dry salt

Dose / conc.:

20 mg/kg bw/day (nominal)

Remarks:

Females /dry salt

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Females / dry salt

Dose / conc.:

0.5 mg/kg bw/day (nominal)

Remarks:

Males & females/dry salt. Satellite groups for systemic organ toxicity assessment (liver)

No. of animals per sex per dose:

Groups of 10 males received the test item, by gavage at dosages of 5, 20 and 60 mg/kg/day (dry salt) and groups of 10 females received the test item at dosages of 5, 20 and 80 mg/kg/day (dry salt).
One satellite group for liver toxicity evaluation, comprised 5 male and 5 female animals and was treated at 0.5 mg/kg/day (dry salt).

Control animals:

yes

Details on study design:

- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on the previous 28-day study (RTC Study no. 82340), performed at 5, 20 and 60 mg/kg/day in the males and 20, 60 and 200 decreased to 100 mg/kg/day by Week 3, in the females. The dose level of the satellite group was selected in order to assess the NOAEL, not found in the previous study due to the presence of toxic effects in the liver at all the dose levels investigated.

Parental animals: Observations and examinations:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the
presence of post-dose reactions.

BODY WEIGHT: Yes
- Time schedule for examinations: Males (Main and Satellite groups) and Females (Satellite group) were weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Females (Main groups) 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 post partum.

FOOD CONSUMPTION :
Food consumption was recorded at weekly intervals whenever possible, by each cage of rats from allocation to pairing. For female animals, food consumption was also recorded on Days 7, 14 and 20 post coitum, starting from Day 0 post coitum and on Day 4 post partum (starting from Day 1 post partum).

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data

ORGAN WEIGHTS:
In males of the main groups: epididymides and testes
In the females of the main groups: ovaries
In males and females of the satellite group: liver

ORGANS FIXATION:
reproductive organs from all animals of the main groups. Liver from the animals of the satellite group.

HISTOPATHOLOGY:
Study list of organs/tissues for histopathological examination (see below)

Oestrous cyclicity (parental animals):

Vaginal smears were taken daily in the morning from the first day of treatment and up to the end of the mating period, until a positive identification of mating was made. The vaginal smear data were examined to determine the following:
a) anomalies of the oestrous cycle;
b) the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined in male parental generation:
- testis weight, epididymis weight

Litter observations:

As soon as possible after parturition (Day 0 or 1 post partum), the total litter size (live and dead) was counted, sexed and examined for external abnormalities. Live pups were individually identified within the litter. All litters were weighed on Day 1 post partum. All litters were examined daily for dead and abnormal pups. The surviving pups were also weighed on Day 4 post partum. All pups found dead were necropsied.

Postmortem examinations (parental animals):

All females killed at term (including total litter loss), were examined for the following:
a) external and internal abnormalities;
b) number of visible implantation sites (for pregnant animals);
c) number of corpora lutea (if detectable).

Postmortem examinations (offspring):

All live pups were killed on Day 4 post partum and examined for the following:
a) external abnormalities;
b) sex confirmation by gonadal inspection.

Statistics:

For continuous variables the significance of the differences amongst group means were assessed by Dunnett's test or a modified t test, depending on the homogeneity of data.
Statistical analysis of non-continuous variables were 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.
Statistical analysis of histopathological findings were carried out by means of the nonparametric Kolmogorov-Smirnov test (main groups only).

Reproductive indices:

Male Fertility index (%) = (number of males which induced pregnancy/Number of males paired)*100
Female Fertility index (%) = (number of pregnant females/number of females paired)*100
Copulatory interval: mean number of days between pairing and mating
Copulatory index (%) = (number of animals mated/number of animals paired)*100

Offspring viability indices:

Pre-birth loss: (Nb of visible implantations - total litter size)*100 / Nb of visible implantation
Pup loss at birth: (total litter size - liver litter size)*100 / total litter size
Cumulative pup loss on day 4
Sex ratios calculated at birth and on day 4

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No signs of toxicological significance were observed in male animals from the control, low and mid-dose groups.
Hunched posture, piloerection and an emaciated appearance were observed in the majority of the high dose males (receiving 60 mg/kg/day), generally from the end of the second week of treatment.
No significant clinical signs were seen in female animals from the control, low and mid-dose groups. Piloerection and rales were observed in individual high dose females (receiving 80 mg/kg/day).
No clinical signs were observed in the satellite group animals.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights of males were similar between groups during the pre-mating phase of the study. Reductions in body weights (from -12% on Day 8 to -28% on Day 22) were seen in the high dose males from pairing to sacrifice and in the high dose females (from -8% to -20%) during post coitum period when compared to controls.
Reduced body weight gain/body weight loss was observed in the high dose males during pairing up to sacrifice. Terminal body weight showed a statistically significant reduction in the mid- and high dose males (-8% and -30%).
Statistically significant reductions in body weight gain (-45%, -76% and -36% on Days 7, 14 and 20, respectively) were seen the high dose females during the gestation period.

In the satellite group treated for 28 days at 0.5 mg/kg/day, animals gained weight over the entire period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

No effects on food consumption were observed in the males and females during the pre-pairing period.
Reduced food consumption was observed in the high dose females during the post-coitum period. (Female data table)

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant reductions were seen in the high dose females during the post-coitum period, consistent with reduced body weight and body weight gain.

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis 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):

Microscopic evaluation was performed on all animals from the control and high dose groups, liver in the satellite groups and all gross abnormalities from all animals.
As potential treatment-related changes were noted in coagulating gland, seminal vesicle and prostate in high dose males and a reduced number of corpora lutea (ovaries) was observed in high dose females, the histopathological examination was extended to the above mentioned organs in the intermediate dose groups (mid-dose group - 20 mg/kg/day and low dose group - 5 mg/kg/day). In addition, histopathological examination was also extended to prostate gland, seminal vesicles and coagulating glands in all control animals.

Main groups
Treatment-related findings were found in the liver, ovaries, coagulating glands, seminal vesicles, prostate and thymus.
The following are details of the treatment-related lesions seen in the high dosed animals.
Specific notes are added in those organs in which a treatment-related effect was observed in the intermediate and low dose groups.
LIVER: treatment-related changes seen in the high dose included diffused minimal to moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia. This was associated with the presence of minimal to mild inflammatory cell foci, single cell hepatocytic necrosis and multifocal hepatocytic necrosis, minimal multifocal accumulation of brownish pigment-laden macrophages, minimal multifocal bile duct hyperplasia and mild nodular hyperplasia. In the intermediate male group (i.e., Group 3), minimal to moderate diffused hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia, was noted. This was occasionally associated with the presence of minimal single cell hepatocytic necrosis.
THYMUS: treatment-related changes seen in the high dose female included diffused minimal to marked cortical atrophy, resulting from loss of the lymphocytes. This change was usually associated with multifocal lymphocytic necrosis. In a single male rat from Group 2, minimal congestion was noted.
COAGULATING GLAND, SEMINAL VESICLE, PROSTATE: treatment-related changes seen in the high dose included diffused minimal to mild reduced secretion, associated with minimal to mild atrophy of the lining glandular epithelium.
OVARIES: treatment-related changes seen only in high dose indicated mild reduction in the number of corpora lutea when compared to the controls.

All other changes are not considered as related to treatment. In particular, a singular case of monolateral testicular atrophy or absence of sperm and fibrosis in epididymides was observed in a low dose group animal. Such changes are known to occur spontaneously in untreated Sprague Dawley rats of the same age or under our experimental conditions.

Satellite group: No treatment related changes were seen at the histopathological examination.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

No significant differences in oestrus cycle, pre-coital interval and copulation plugs were observed between treated and control animals.
Reproductive parameters (copulatory index and fertility index) did not show intergroup differences in neither sex.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

Absence of sperm and fibrosis in epididymides was observed in a low dose group animal and was considered incidental.

Reproductive performance:

no effects observed

Description (incidence and severity):

No significant differences in pre-coital interval and copulation plugs were observed between treated and control animals.
Reproductive parameters (copulatory index and fertility index) did not show intergroup differences in neither sex.

All females were pregnant. Unilateral implantation was recorded in 1 female of the control group. All females mated, even though mating was not detected in 1 mid-dose female.

Reductions in the number of implantation and corpora lutea (-17% and -13%, respectively, although not statistically significant) were observed in the high dose dams (80 mg/kg/day). Total litter size was significantly reduced in these animals. Due to the mortality or cannibalisation of all pups between Days 0 and 1, pre-birth loss number and percentage were not calculable in Group 4 animals.
No differences in the number of implantation or pre-birth loss data were noted in the other treated animals compared to controls.
No significant differences in gestation length were observed. The slight reduction observed in animals of Group 3 was not considered of toxicological relevance.

Males were more sensitive to liver effects than females (effects observed at a lower dose).
In males of the high dose (60 mg/kg/day), effects consisted of reduced body weight, Increased relative weights of testes, various histopathological findings in the liver (including hepatocytic hypertrophy, inflammation, hepatocytic necrosis, bile duct hyperplasia). Treatment-related findings were found in the coagulating glands, seminal vesicles, and prostate. Milder effects were noted in the intermediate dose group.
In females of the high dose group (80 mg/kg/day), effects consisted of reduced body weight, reduced food consumption during post-coitum period; macroscopic changes in the liver and thymus, histopathological findings in the liver, thymus, and ovaries. Females of this group had a lower number of corpora lutea. Iin addition, there was a reduction in the number of implantations and corpora lutea, and a peri-natal litter loss.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 20 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

other: females

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

60 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

other: males

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Pre-weaning clinical signs observed in the pups during the lactation period showed small pups, often showing an apparent absence of food intake, with scabs or bruises on the muzzle. The incidence of these signs increased with the dose level of dams.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The number of females with live pups on Day 4 post partum was 10 in each of the control, low dose and mid-dose groups. Total litter loss or cannibalisation during parturition were observed on Days 0 or 1 post partum in the high dose dams which were sacrificed.

All pups of high dose dams were found dead or were cannibalised between Days 0 and 1. Those which could be observed showed an apparent absence of food intake.
Necropsy examination of the early decedent pups revealed the absence of milk in the stomach in the majority of pups from all dose groups. Autolysis was seen in a number of pups from all dose groups.
No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects were observed on the mean total and individual litter weights in the 2 lowest dose groups.
The high dose group could not be assessed due to total litter loss in all dams.

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:

effects observed, treatment-related

Description (incidence and severity):

All pups of high dose dams were found dead or were cannibalised between Days 0 and 1. Those which could be observed showed an apparent absence of food intake.
Necropsy examination of the early decedent pups revealed the absence of milk in the stomach in the majority of pups from all dose groups. Autolysis was seen in a number of pups from all dose groups.
No significant abnormalities were recorded in the pups sacrificed on Day 4 post partum.

All pups from Group 4 litters (dams dosed at 80 mg/kg/day) were found dead or were cannibalised during parturition or between the day of parturition (Day 0) and Day 1. Those which could be observed showed an apparent absence of food intake.
Total litter size showed a significant reduction in animals dosed at 80 mg/kg/day, when compared to controls (-50%). Live litter size of the high dose group was also consequently severely reduced on Day 0, with total litter loss recorded on Day 1 post partum. The mortality or cannibalisation of all pups from the high dose dams did not allow an evaluation of mean total and individual litter weights or the sex ratio.

Total litter size, live litter size and pup loss were comparable between Groups 2, 3 (dosed at 5 and 20 mg/kg/day, respectively) and controls on Days 1 and 4 post partum. A slight but not statistically significant reduction (-8%) in mean pup weight was seen in animals from mid-dose groups (20 mg/kg/day). This slight reduction was not considered to be of toxicological relevance as, in the majority of cases, parturition occurred on Day 21.
Sex ratio (as % of males) and the total number of pups were comparable between Groups 2 and 3 and controls on Days 1 and 4 post partum.

Since the indicated effects on litters noted in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the developmental effects could not be done at this dose level.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 20 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

other: At the high dose, smaller litter size due to perinatal loss or cannibalism during parturition or between day 0 and 1.

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Since the indicated effects on pregnancy noted in the high dose females were observed in the presence of a marked maternal toxicity a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.

Reproduction data - summary

Dose (mg/kg/day)	0	5	20	80
Females paired, N	10	10	10	10
Females mated, N	10	10	10	10
Pregnant females, N	10	10	10	10
Mating index, %	100	100	100	100
Fertility index, %	100	100	100	100
Mean pre-coital time, days	5.9 + 4.5	3.2 + 1.5	3.7 + 2.4	4.4 + 3.6
Mean corpora lutea, N	15.80 + 3.61	15.60 + 1.71	15.90 + 2.56	13.70 + 2.83
Mean implantation sites, N	15.50 + 3.84	15.20 + 2.25	15.80 + 2.49	12.80 + 4.02
Gestation length, days	22.00 + 0.00	21.80 + 0.42	21.44 + 0.53	21.90 + 0.57
Litter number, N	10	10	10	(6)	high dose: ND due to cannibalism at parturition
Mean total litter size, N	14.60 + 4.17	14.90 + 2.28	14.80 + 2.78	°
Mean pre-birth loss, N	0.90 + 0.74	0.30 + 0.48	1.00 + 1.05	°
Pre-birth loss, %	6.59 + 5.94	1.97 + 3.17	6.54 + 7.06	ND

° not included in statistical analysis

ND not determinable

Litter data - summary (groups 1 to 3)

Dose (mg/kg/day)	0	5	20
Litter number, N	10	10	10
Total litter size at birth, mean	14.60 + 4.17	14.90 + 2.28	14.80 + 2.78
Live pups at birth, mean	14.30 + 4.08	14.60 + 2.12	14.60 + 2.76
Pup loss, %	1.88 + 4.04	1.86 + 3.01	1.36 + 2.95
Litter affected, N	2	3	2
Total litter size at birth, N	146	149	148
Total live pups at birth, N	143	146	143
Day 1 post-partum:
Mean litter weight day 1 p.p., g	84.81 + 26.72	89.70 + 12.73	82.54 + 14.19
Mean pup weight day 1 p.p., g	6.38 + 0.25	6.18 + 0.46	5.84 + 0.53
Day 4 post-partum:
Mean live litter size on day 4, g	13.50 + 3.60	14.00 + 2.00	13.70 + 2.21
Cumulative loss on day 4, %	6.51 + 8.11	5.43 + 8.98	6.85 + 7.02
Litter affected, N	5	5	7
Total litter size on day 4 p.p., N	135	140	137
Mean litter weight day 4 p.p., g	113.3 + 24.40	119.4 + 20.51	102.4 + 13.80
Mean pup weight day 4 p.p., g	8.55 + 1.15	8.56 + 0.94	7.57 + 1.04
Sex ratio:
Sex ratio at birth, % males	52.30 + 20.76	49.82 + 12.47	53.39 + 18.85
Sex ratio on day 4 p.p., % males	52.45 + 20.11	50.25 + 12.88	54.85 + 19.41

Conclusions:

On the basis of the above results, treatment-related effects on pregnancy and on early lactation of the offspring, were observed in dams dosed at 80 mg/kg/day. Some treatment-related effects (as minor clinical signs, reductions in body weight) were also noted during the mating and gestation periods in the high dose males and females.
Reproductive parameters such as fertility index, pre-coital interval and copulatory index were not affected by treatment in neither sex at any dosage. The gestation length was not altered.

Adverse effects were seen in the high dose females (reductions in the number of implantation sites and of corpora lutea) and at parturition and total litter loss by cannibalism by day 1. Since these effects in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.

No adverse effects on reproduction and pup development were observed in animals dosed at 20 mg/kg/day or 5 mg/kg/day. However, minimal to moderate effects on liver were observed at microscopic examination in some animals dosed at 20 mg/kg/day (parental males).
The NOAEL was set at 20 mg/kg/day for reproductive parameters and development.

Executive summary:

The purpose of this study was to investigate the effects of the test substance on reproduction, in 10 male and 10 female rats and on the development of offspring, when treated for at least 2 weeks before mating and throughout the gestation and lactation periods, until Day 3 post partum. Males received doses of 0, 5, 20 and 60 mg/kg/day, and females received doses of 0, 5, 20, and 80 mg/kg/day.

The potential liver toxicity was also evaluated at a lower dose of 0.5 mg/kg/day in a satellite group of 5 males and 5 females.

The study design was based on the procedures described in OECD guideline number 421, “Reproduction/ Developmental Toxicity Screening Test” (1995) and performed in compliance with GLP.

 

Minor clinical signs (piloerection, occasional rales) were detected in the high dose males and females from the third week in vivo phase (mating phase). Reductions in body weight/body weight gain and food consumption were observed in males and females from this dose group starting from the mating phase. Statistically significant reductions of body weight were still present in the high dose females during gestation period and in the males up to termination. Some effects (minor clinical signs, reductions in body weight/ body weight gain) were also noted during the mating period in the high dose males and females and during the gestation period in the high dose females.

At macroscopic and microscopic examination, treatment-related findings were found in the liver, coagulating glands, seminal vesicles, prostate, ovaries and in the thymus of the high dose animals. No treatment-related changes were seen in the testes. Reproductive parameters such as fertility index, pre-coital interval and copulatory index were not affected by treatment in neither sex at any dosage.

Adverse effects seen in the high dose females consisted of reductions in the number of implantation sites and of corpora lutea. At parturition, litters were lost by cannibalism or were smaller in size and weight, before total loss by day 1. Reductions in mean total and individual litter weights (when calculable) were the main treatment-related effects observed in dams dosed at 80 mg/kg/day in this study.

Since these effects in the high dose females were observed in the presence of a marked maternal toxicity (severe reductions in body weight and body weight gain, severe changes in the liver and thymus) a correct evaluation/interpretation of the reproductive effects could not be done at this dose level.

No significant effects were observed in animals dosed at 20 mg/kg/day. The slight reduction in mean pup weight seen on Day 1 was not considered to be significant as the mean total litter weight was not significantly reduced and, in the majority of cases, parturition occurred on Day 21 (not on Day 22 as in the controls). No effects on fertility, pregnancy and early lactation of the offspring were observed in animals dosed at 5 mg/kg/day. At macroscopic and microscopic examination no treatment-related lesions were seen in animals treated at 5 and 20 mg/kg/day. However, effects on liver were observed at microscopic examination in some parental animals dosed at 20 mg/kg/day. No treatment related changes were seen at the histopathological examination of the liver performed in satellite group animals, dosed at dosed at 0.5 mg/kg/day for 28 consecutive days.

Based on the results of this study, the NOAEL for reproductive toxicity and development is set at 20 mg/kg/day.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

20 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

GLP-compliant study with Klimisch 1

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

In a GLP-compliant prenatal developmental toxicity study performed according to OECD Guideline 414, the NOAEL was considered to be 60 mg/kg bw/day for maternal toxicity based on swollen and/or enlarged liver, and 80 mg/kg bw/day for developmental toxicity in Sprague-Dawley rats based on the absence of fetal malformations.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Remarks:

(The study was performed to comply with a Regulation outside Europe)

Adequacy of study:

key study

Study period:

From 10-APR-2013 to 05-JUN-2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 414 without major deviation.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italy S.p.A., Calco (Lecco), Italy.
- Age at study initiation: 9 weeks old for females, 11 weeks for males.
- Weight at study initiation: 204-234 g for females, at least 340 g for males.
- Housing: The animals were housed in a limited access rodent facility. During the pre-pairing period and after mating, the animals were housed no more than 5 of one sex to a cage in Polysulfone cages measuring 59.5x38x20 cm. During the mating period, the rats were housed on the basis of 1 male to 1 female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor (Techniplast Gazzada S.a.r.l., Buguggiate, Varese).
- Diet: 4 RF 21 (Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy) laboratory rodent diet, ad libitum
- Water: drinking water supplied via water bottles, ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 55 ± 15 %
- Air changes: approximately 15 to 20 per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 30-April-2013 To: 29-May-2013

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS :
On a daily basis, a sufficient amount of the test item was diluted 1:10 with purified water, to obtain a concentration of 39.8 mg/mL (stock solution). Then further independent solutions were prepared with the same vehicle from the stock solution to give the required concentrations of 0.5, 2, 6 and 8 mg/mL. Concentrations were calculated and expressed in terms of dry salt (39.8 %).

VEHICLE
- Concentration in vehicle: 0.5, 2, 6 and 8 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight

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 (content check). Samples of the formulations prepared on Week 1 and last week were also analysed to check the concentration.
- Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study nos. 82340, 82350 and 89720) in the range from 0.03 to 100 mg/mL in purified water.

Results: Results of all analyses were within the limits of acceptance.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1; Females were paired one to one in the home cage of the male and left overnight. Vaginal smears were taken daily in the morning, from the day after pairing until a positive identification of mating was made.
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: not applicable
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy (or Day 0 post coitum)

Duration of treatment / exposure:

14 days (Days 6-19 post coitum)

Frequency of treatment:

Once daily, from Day 6 through Day 19 post coitum

Duration of test:

21 days (Days 0-20 post coitum)

No. of animals per sex per dose:

24 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels of 5, 20, 60 and 80 mg/kg bw/day were selected in agreement with the Sponsor, based on information from previous studies.
- Rationale for animal assignment: Before the beginning of the treatment period, the animals were allocated to the groups, according to a computerised stratified randomisation based on body weight recorded on Day 0 post coitum, to give approximately equal initial group mean body weights.

Maternal examinations:

CAGE SIDE & CLINICAL OBSERVATIONS: Yes
Time schedule:
- Mortality or signs of morbidity: Twice a day throughout the study, including weekends and public holidays.
- Clinical signs: Once daily. Clinical signs for one animal were erroneously not recorded on Day 1 post coitum.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 3, 6, 9, 12, 15, 18 and 20 post coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Days 3, 6, 9, 12, 15, 18 and 20 post coitum, starting from Day 0 post coitum.

POST-MORTEM EXAMINATIONS: Yes
- Time schedule: On Day 20 post coitum, females were sacrificed by inhalation of carbon dioxide and were subjected to a macroscopic post-mortem examination which included the observation of all visible structures, surfaces and orifices.

OTHER:
Preservation of tissues:
- Changes were noted and the abnormalities preserved in 10% neutral buffered formalin.
- From all adult females completing the scheduled test period, liver was preserved in 10% neutral buffered formalin.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes, , uteri or individual uterine horns without visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of embryonic death at very early stages of implantation
- Number of late resorptions: Yes
- Other: uterine scars, gross evaluation of placentas

Fetal examinations:

EXAMINATION OF FETUSES:
- Number of all live fetuses: Yes
- Number of dead fetuses (fetuses at term without spontaneous movements and breathing): Yes
- Live fetuses were sacrificed by intraperitoneal injection of Sodium Thiopental followed by hypothermia.
- Body weight of fetuses: Body weight of each live fetus was recorded.
- Sex of fetuses: Sex of each fetus was determined at the time of caesarean.
- External examinations: Yes; each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices.
- Soft tissue examinations: Yes, approximately half of the live fetuses in each litter were eviscerated and were fixed in Bouin’s solution.
- Skeletal examinations: Yes [all the remaining litters]; eviscerated and fixed in 95% (v/v) ethanol, for subsequent skeletal examination.
- Head examinations: No data

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.
- Statistical analyses of non-continuous variables were 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 and statistical analysis were calculated from actual values in the computer without rounding off.

Indices:

- Pre-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] X 100
- Post-implantation loss: [(Number of implantation sites - Number of live fetuses) / Number of implantation sites] X 100
- Total impl antation loss: [(Number of corpora lutea - Number of live fetuses) / Number of corpora lutea] X 100
- Sex ratios of the foetuses were calculated as the percentage of males per litter.
- All derived values (e.g., means, percentages, ratios) were first calculated within the litter and the group values derived as a mean of individual litter values.
- Foetal structural deviations were expressed as the percentage of affected foetuses relative to all foetuses examined per group, as well as in terms of the mean litter percentage of affected litters.

Historical control data:

No data

Clinical signs:

no effects observed

Description (incidence and severity):

No relevant clinical signs or signs of reaction to treatment were noted in treated females.
Hairloss was observed in few females of the control and treated groups. In addition, some of these females, with the exception of those of the high dose groups, showed also scabs. Vaginal discharge was observed in one female receiving 60 mg/kg/day on Day 15 post coitum.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- No differences in body weight were noted between control and treated groups.
- On Day 9 post coitum, a decrease in body weight gain between -4% to -26% was recorded in all treated groups when compared to controls, with a statistical significance in mid-, mid-high and high dose groups.
- In addition, a decrease, between -21% to -33% in body weight gain, was noted in females receiving 60 and 80 mg/kg/day on Day 12 post coitum, in females receiving 60 mg/kg/day on Day 15 post coitum and in females receiving 5 mg/kg/day on Day 20 post coitum.
- Recovery was observed on Day 18 post coitum, where an increase in body weight gain was recorded in all treated females when compared to controls.

Terminal body weight, uterus weight and absolute weight gain of females
- No significant differences in terminal body weight and gravid uterus weight were observed in treated groups when compared to the control group. The statistically significant decrease in absolute weight gain detected in midhigh (-18%) and high dose (-13%) groups with respect to controls was not considered of toxicological relevance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

- The statistically significant decrease up to approximately 11%, detected on Days 12 and 15 post coitum in females receiving 60 and 80 mg/kg/day, was not considered of toxicological relevance.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No differences of toxicological relevance were noted in gravid uterus weight between treated and control groups (table 7.8.2-3)

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Maternal necropsy findings:
- The only relevant change detected at post mortem examination was swollen and/or enlarged liver in some females dosed at 80 mg/kg/day indicative of maternal toxicity at this dose level.
- The remaining macroscopic changes, such as swollen liver in few females dosed at ≥ 20 mg/kg/day, pale area/s in the left lobe of the liver in single females dosed at 60 or 80 mg/kg/day or marked hairloss mainly noted in few females dosed at 60 mg/kg/day, could be considered incidental and not clearly treatment related.

Histopathological findings: non-neoplastic:

not examined

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

The lower absolute weight gain noted at the doses 60 (-18%) and 80 (-13%) mg/kg/day compared to the control group was not considered of toxicological relevance. The differences were likely related to the slightly higher gravid uterus weights in those 2 groups. (Table 7.8.2-3)

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

No statistically significant differences were observed between groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

The number of females with live foetuses on Day 20 post coitum was 24 in each of the control and treated groups.
One foetus was found dead in the lowest dose group.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

All females were found pregnant at necropsy.
One control female and one female receiving 5 mg/kg/day had unilateral implantation.

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

gross pathology

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

other: Liver

Description (incidence and severity):

4/24 females at the highest dose displayed enlarged liver, and 2/24 had swollen liver.

Fetal body weight changes:

no effects observed

Description (incidence and severity):

mean foetal weight was not affected by treatment.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

The mean number of viable foetuses was similar in all groups

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

Litter data were not affected by treatment: mean litter size and mean litter weight were similar in all groups

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External observations:
- The malformation imperforation of anus was observed in one foetus of the mid-high dose group and one dead foetus was detected in the low dose group.
- A total of 11 small foetuses (<2.7 g) were seen: one out of 342 in control group, one out of 348 in low dose group, three out of 352 in mid-dose group and six out of 370 in mid-high dose group. No small foetuses were noted in the high dose group.
- All these findings were not dose-related and therefore considered incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal and cartilage observations:
- A total of 9 foetuses showed malformations at skeletal examination: 4 in the control group, 2 in the mid-dose group, 3 in the mid-high dose group. No malformations were detected in the low and high dose groups.
- Lumbar hemivertebrae, pubis no ossification, ribs fused or displaced were the malformations detected in 4 different litters of the control group. One of these litter also showed a foetus with dextrocardia at visceral examination.
- Pubis no ossification and absence of one rib were the malformations detected in 2 different litters of the mid-dose group.
- Centra fused of a lumbar vertebrae, pubis no ossification and absence of one rib were the malformations detected in 2 different litters of the mid-high dose group. One of these females also showed a foetus with malformation at visceral examination.
- Other alterations recorded at skeletal examination were noted both in control and treated groups, with a similar incidence.
- Considering that some of these findings were observed in small foetuses (foetal weight less than 2.7 g) and were without a dose-relation, they were considered incidental and not related to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue observations:
- A total of 4 foetuses showed malformations at visceral examination: 1 (dextrocardia) in the control group, 2 (anophtalmia and hydrocephalia) in the low dose group and 1 (agenesis of ovaries and malpositioned uterus) in the mid-high dose group. No malformations were detected in the mid- and high dose groups.
- Due to the absence of a dose-relation and to the low incidence of malformations, variations and anomalies, all the findings detected at visceral examination were considered incidental.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 80 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Incidental findings detected at the external, visceral and skeletal examination of foetuses from all groups with no dose-relationship.

Key result

Abnormalities:

effects observed, non-treatment-related

Description (incidence and severity):

Incidental findings detected at the external, visceral and skeletal examination of foetuses from all groups with no dose-relationship.

Key result

Developmental effects observed:

no

MATERNAL DATA

Table 7.8.2-2: Mean maternal body weight gain (g)

Dose (mg/kg/day)	0	5	20	60	80
GD 3	7.243	6.064	7.240	7.209	6.180
GD 6	5.948	6.234	6.150	6.294	6.674
GD 9	5.816	5.555	4.650*	4.290**	4.654*
GD 12	6.948	6.266	7.191	5.328**	4.667**
GD 15	6.358	7.453	6.269	5.025*	5.267
GD 18	12.498	14.705*	14.901*	14.570	15.394**
GD 20	19.185	14.475*	17.353	17.859	18.302

 GD: gestation day; *: p<0.05; **: p<0.01

Table 7.8.2-3: Terminal body weight, uterus weight and absolute weight gain (g) - group mean data (24 females/group)

Dose (mg/kg/day)	0	5	20	60	80
Terminal body weight (g)
mean	410.50	410.82	411.64	399.50	405.05
sd	28.41	27.61	23.08	29.06	28.42
Gravid uterus weight (g)
mean	84.12	85.90	86.73	88.72	87.28
sd	18.24	16.72	8.36	9.32	10.99
Absolute weight gain (g)#
mean	83.17	82.79	82.15	67.90*	72.46*
sd	16.29	16.69	14.20	16.07	13.61

# body weight at necropsy minus gravid uterine weight, minus body weight at day 0 of pregnancy

Statistical analysis: Krukall Wallis test, or William's test if group differences are different from control at p< 0.05

*: p< 0.05

Tablea 7.8.2-4: Main macroscopic findings in females

Dose (mg/kg/day)	0	5	20	60	80
N examined	24	24	24	24	24
Liver
abnormal area(s), pale	0	0	0	1	1
Abnormal shape, swollen	0	0	3	2	2
abnormal size, enlarged	0	0	0	0	4
Uterus
unilateral implantation	1	1	0	0	0

No microscopic examination was performed in this study

Conclusions:

There were no treatment-related effects on maternal parameters (corpora lutea, implantation sites, intra-uterine deaths) or on foetal data (live foetuses, litter or foetus weights, sex ratio or foetal development). Under the test conditions, the No Observed Adverse Effect Level (NOAEL) of cC6O4 was considered to be 60 mg/kg bw/day for maternal toxicity (based on macroscopic findings and lower absolute weight gain) and 80 mg/kg bw/day (highest tested dose level) for developmental toxicity in Sprague-Dawley rats.

Executive summary:

In a GLP-compliant prenatal developmental toxicity study performed according to OECD Guideline 414, cC604 diluted in water was administered by gavage to groups of mated female Sprague-Dawley rats (24 mated females/dose) at the dose levels of 0, 5, 20, 60 and 80 mg /kg bw/ day (in terms of dry salt (39.8 %)) from Days 6 to 19 post-coitum. Clinical signs and mortality were checked daily. Maternal body weight and food consumption were recorded approximately every 3 days. On Day 20 post-coitum, the dams were sacrificed and subjected to macroscopic examination. The gravid uterine weight, number of implantations, uterine scars, live and dead fetuses, early and late resorptions and corpora lutea were recorded. Gross evaluation of the placenta was also performed. Fetuses were sexed, weighed and examined for external, soft tissue and skeletal malformations.

All mated animals were pregnant. No mortality was observed. No relevant clinical signs or signs of reaction to treatment were noted in treated females. No differences of toxicological relevance were noted in body weight, food consumption, gravid uterus weight and litter data of treated females when compared to controls. Swollen and/or enlarged liver was detected at macroscopic examination in some females dosed at 80 mg/kg/day, indicating the presence of a maternal toxicity at this dose level.

Due to the absence of a dose-relation, the findings detected at the external, visceral and skeletal examination of foetuses from all groups were considered to be incidental.

On the basis of the results obtained in this study, the dosage of 60 mg/kg bw/day was considered to be the NOAEL (No Observed Adverse Effect Level) for maternal toxicity. The NOAEL for developmental toxicity was considered to be 80 mg/kg bw/day.

This study was considered as acceptable as it satisfied the main criteria of the OECD guideline.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a GLP-compliant prenatal developmental toxicity study performed according to OECD Guideline 414, the test substance diluted in water and administered by gavage to gestating Sprague Dawley rats at doses up to 80 mg /kg bw/ day from Day 6 to Day 19 post-coitum did not induce any significant malformation or variation in the fetuses, and did not induce any dose-related changes in the pregnancy parameters (i.e., mean number of corpora lutea, implantation sites, intra-uterine deaths, live fetuses, sex ratio, litter and fetal body weights). Swollen and/or enlarged liver was detected at macroscopic examination in a few females dosed at 80 mg/kg/day, indicating the presence of a maternal liver toxicity at this dose level.

Therefore, maternal NOAEL was considered to be 60 mg /kg bw/ day, and the NOAEL for prenatal development was considered to be 80 mg/kg bw/day.

Toxicity to reproduction: other studies

Additional information

In the Reproduction/Developmental Toxicity Screening study (OECD421), dams dosed at 80 mg/kg bw/day (highest dose group) showed treatment-related effects on pregnancy and on early lactation of the offspring. Treatment-related effects (as minor clinical signs, significant reduction in body weight and body weight gain, reduced food consumption) were also noted during the mating and gestation periods in the high dose females (80 mg/kg bw/d), as well as in high dose males (60 mg/kg/d).

Reproductive parameters such as estrus cycle, fertility index, pre-coital interval and copulatory index were not affected by treatment in neither sex at any dosage. The gestation length was not altered.

There was a reduced mean number of implantations and corpora lutea in the female high dose group (not statistically significant).

At macroscopic and microscopic examination of the high dose parental animals, treatment-related findings were observed in the liver (males, females), and were consistent with effects seen in an earlier 28-day oral repeated dose study. In particular, the main target organ (liver) was swollen, enlarged and pale or dark aspect. At microscopic observation, treatment-related changes included diffused minimal to moderate hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia.

This was associated with the presence of minimal to mild inflammatory cell foci, single cell hepatocytic necrosis and multifocal hepatocytic necrosis, minimal multifocal accumulation of brownish pigment-laden macrophages, minimal multifocal bile duct hyperplasia and mild nodular hyperplasia.

Some males dosed at 20 mg/kg bw/day had also microscopic lesions on the liver, with a lower severity

Atrophy of the coagulating glands, seminal vesicles, and prostate was observed in the high dose group males, but no treatment-related changes were seen in the testes. The high dose group females had a mild reduction of corpora lutea in the ovaries, and a minimal to marked thymus atrophy.

No significant effects on reproduction as well as on fertility were observed in animals dosed at 20 mg/kg bw/day and no effects on fertility, pregnancy and early lactation were observed in animals dosed at 5 mg/kg/day.

The 28-day oral repeated dose toxicity study provides additional information relevant for the evaluation of the adverse effects in the high dose females. Groups of females in the 28-day received doses below (60 mg/kg/d) and above (200/100 mg/kg/d) the highest dose for females in the OECD421 study. Besides the marked hepatic toxicity, various changes in these groups of non-pregnant females are consistent with general toxicity and markers of a systemic stress response (Everds et al., 2013): reduction in body weight (-23%), body weight gain, food consumption, significant decrease in the relative weight of organs such as ovaries (-23.6%), thymus (-2.5%), and spleen (-25.5%), associated with thymus and spleen atrophy and decreased eosinophils. However, the relative weight of adrenal glands was also decreased in these females. Effects seen in males of the high dose group (60 mg/kg/d) also support a stress response: reduction in body weight, body weight gain, food consumption, changes in hematology parameters, increased weights (absolute and relative) of adrenal glands, while reduced weights (absolute and relative) of thymus, spleen, and accessory sex organs (epididymides, prostate), were associated with atrophy at microscopic examination.

Based on the observations from the 28-day toxicity study, the litter loss in the high dose group between day 0 and day 1 could be related to maternal toxicity. The pups that could be assessed in this group showed no milk intake, further indicative of lack of maternal care.

The main adverse effects in the 90-day study on organ weights and histopathology were observed in the liver, further supporting a non-specific toxicity at the doses above 10 mg/kg/day, and that the NOAEL from the repeated dose toxicity is protective from potential adverse effects on reproduction and development.

Justification for classification or non-classification

Adverse effects on sexual function and fertility:

Reproductive parameters such as fertility index, pre-coital interval and copulatory index were not affected by treatment in either sex, at any dosage.

The effects on early postnatal development could not be fully assessed due to the loss of the litters by cannibalisation of pups. The concomitant signs of significant general systemic toxicity in the dams treated at the highest dose indicate this behaviour and lack of maternal care could be related to maternal toxicity and systemic stress. No significant developmental effects were noted in the pups of the lower doses, in the absence of obvious maternal toxicity.

 

Adverse effects on development of the offspring:

As the substance did not induce any effects on fetal developmental in a GLP study conducted according to OECD guidelines 414, it is not classified for developmental toxicity according to the Annex VI of the Directive 67/548/EEC and to the CLP Regulation (EC) N° 1272/2008. 

 

Conclusion

Based on the results of a reproductive toxicity screening test and a pre-natal developmental toxicity study, the substance does not need classification as a reproductive toxicant.