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Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

Reproductive Toxicity Study:

Based on all the available observations and results, it was concluded that the test chemical does not cause any defects in any the reproductive functions and parameters even when it is exposed at high dose levels and therefore is not likely to be classified as a 'reproductive toxicant' as per the CLP criteria of classification and labelling.

Link to relevant study records
Reference
Endpoint:
fertility, other
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
data from handbook or collection of data
Justification for type of information:
data is from safety assessment reports
Qualifier:
according to guideline
Guideline:
other: as mentioned below
Principles of method if other than guideline:
To determine the effects of maternal exposure to the test chemical during prenatal development phase
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
not specified
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeding colony (Imp: DAK).
- Females (if applicable) nulliparous and non-pregnant: [yes/no] : yes, Nulliparous female rats
- Age at study initiation: (P) x wks; (F1) x wks : Nulliparous female rats, aged approximately 14 weeks
- Weight at study initiation: (P) Males: x-x g; Females: x-x g; (F1) Males: x-x g; Females: x-x g : Nulliparous female rats, aged approximately 14 weeks, weighing 199 + 12 g
- Fasting period before study:
- Housing: quarters
- Use of restrainers for preventing ingestion (if dermal): yes/no
- Diet (e.g. ad libitum): The animals were maintained on commercial pelleted chow (Fodder Factory, Motycz, Poland), ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: no daata available

ENVIRONMENTAL CONDITIONS
- Temperature (°C): controlled temperature (approx. 22°C)
- Humidity (%): Relative humidity varied between 45 — 55%.
- Air changes (per hr): no data available
- Photoperiod (hrs dark / hrs light): 12 hours cycle, illumination (light on between 6 a.m. and 6 p.m.)
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the inseminated females were divided into one control and three treatment groups receiving 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier found to be 5.8 g/kg.
Details on mating procedure:
- M/F ratio per cage: Females were mated overnight with 17-week-old male rats of the same strain.
- Length of cohabitation: Females were mated overnight with 17-week-old male rats of the same strain.
- Proof of pregnancy: [vaginal plug / sperm in vaginal smear] referred to as [day 0 / day 1] of pregnancy: The morning when copulation plugs or spermatozoa appeared in
vaginal smears was regarded as day 1 of gestation
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
The test chemical was administered orally, by gavage, every other day from day 8 do day 20 of gestation.
Frequency of treatment:
daily
Details on study schedule:
no data available
Remarks:
Prenatal Phase study:0, 140, 580, 1150 mg/kg.day
Basis: 0.025, 0.1 and 0.2% of median lethal dose of the test chemical, LD50 = 5800 mg/kg
No. of animals per sex per dose:
exact number of rats not mentioned
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the inseminated females were divided into one control and three treatment groups receiving 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(5800 mg/kg).
Positive control:
no data available
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data: YES
- Time schedule: DAILY
- Cage side observations checked in table [No.?] were included. : General appearance of the dosed rats was observed daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data: yes
- Time schedule: daily

BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations: The maternal weight gain was observed throughout the gestation period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data: The daily food and water
consumption intake were monitored throughout the gestation period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: no data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data: yes
- Time schedule for examinations:The daily water consumption intake were monitored throughout the gestation period.
Oestrous cyclicity (parental animals):
no data available
Sperm parameters (parental animals):
no data available
Litter observations:
PARAMETERS EXAMINED
The following parameters were examined in [F1 / F2 / F3] offspring:
[number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention should be paid to the external reproductive genitals which should be examined for signs of altered development; gross evaluation of external genitalia] : The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded. A site was judged as a late resorption when macroscopic discrimination between fetal residues and placental material was possible. When this discrimination could not be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each female. Live fetuses were measured for body weight, crown-rump length and examined for external malformations.

GROSS EXAMINATION OF DEAD PUPS:
[no / yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead] :
Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals [describe when, e.g. as soon as possible after the last litters in each generation were produced.]
- Maternal animals: All surviving animals [describe when, e.g. after the last litter of each generation was weaned.]: The female rats were killed on day 21 of
gestation under ethyl ether anesthesia

GROSS NECROPSY
- Gross necropsy consisted of [external and internal examinations including the cervical, thoracic, and abdominal viscera.]: The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table [#] were prepared for microscopic examination and weighed, respectively.
Statistics:
In the case of homogeneity of variance, one-way analysis of variance and Dunnett’s test were used: in the case of heterogeneity the Kruskal-Wallis analysis of variance was followed by non-parametric tests. Frequency data were analyzed with Fisher’s exact probability test. The effect of test chemical on behavioral performance, food and water consumption and body weight gain of the offspring was evaluated with two-way analysis of variance and Scheffe’s
test for multiple comparison.
Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behaviour of the exposed and control animals did not differ significantly
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The toxic effect of the test chemical on the organism of the pregnant female rats was manifested by significantly smaller body weight gain during their pregnancy
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The daily intake of the food of the exposed and control animals did not differ significantly.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The daily intake of the water of the exposed and control animals did not differ significantly.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
the general appearance and behaviour of the exposed and control animals did not differ significantly.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Female rat fertility in terms of the percentage of the inseminated female rats found to be pregnant, and female rat fecundity in terms of the number of live fetuses in a litter were similar in the control group and in the groups exposed to the test chemical
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
The test chemical when administered to the maternal rats at the dose of 1.15 g/kg (0.2 LD50) showed fetotoxic effect, causing a reduction of fetus body weights and length. The weight of the placenta in the female rats poisoned with 0.14 g/kg - 1.15 g/kg of this chemical was significantly higher than that in the controls. The dose-effect relationship, however, was not detected
Dose descriptor:
NOAEL
Effect level:
580 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
reproductive function (oestrous cycle)
reproductive performance
Critical effects observed:
no
Clinical signs:
not specified
Dermal irritation (if dermal study):
not specified
Mortality / viability:
no mortality observed
Description (incidence and severity):
The number of live and dead litter per dose group didnot differ significantly from the controls
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The test chemical when administered to the maternal rats at the dose of 1.15 g/kg (0.2 LD50) showed fetotoxic effect, causing a reduction of fetus body weights and length.
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Sexual maturation:
not specified
Anogenital distance (AGD):
not specified
Nipple retention in male pups:
not specified
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macro evaluation of the internal organs of the fetuses did not reveal any macro pathology within the organs in any of the evaluated groups of the animals
Histopathological findings:
not specified
Other effects:
effects observed, treatment-related
Description (incidence and severity):
The frequency of occurrence of the delays in epicranial bones and sternum ossification was significantly higher in the groups which were prenatally exposed to higher doses of the test chemical
Developmental immunotoxicity:
not specified
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
580 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
Critical effects observed:
no
Reproductive effects observed:
no

Table 1. Absolute and relative organ weights of female rats receiving daily by gevage test chemical on days 8—20 of gestation.

 

Control

0.14 g/kg

0.58 g/kg

1.15 g/kg

Liver

14

18

18

17

(g)

11.69 ± 1.5“

 11.68 ± 1.27

 11.62 + 1.08

 11.21 + 1.02

(g%)

3.91 + 0.24

 3.86 ± 0.24

3.87 + 0.24

3.85 + 0.23

Kidneys

14

18

18

17

(g)

1.36 + 0.15

1.40 ±0.11

1.37 ± 0.13

1.37 ± 0.12

(g%)

0.46 ± 0.03

0.47 + 0.04

0.46 ± 0.03

0.47 ± 0.04

Adrenals

14

18

18

17

(mg)

71.4 + 6.3

72.9 + 9.0

 74.4 ± 8.2

80.1 ± 13.8

(mg%)

24.2 + 2.6

 24.3 ± 3.8

24.9 ± 2.7

27.7 ± 5.9*

 

Table 2: Effect of test chemical administered within 8 — 20 days of gestation on pregnancy and development in rats

 

Control

0.14 g/kg

0.58 g/kg

1.15 g/kg

Females inseminated

17 (82.4)a

 19 (94.7)

18 (100)

19 (89.5)

Live fetuses per litter

11.8 ± 2.2 b

11.3 ± 2.2

11.1 ± 1.6

12.0 ± 2.0

Dead fetuses per litter

0

0

0.06 ± 0.24

0.12 ± 0.33

Litters with resorptions

3

10

9

7

Litters with late resorptions

2

6

4

4

Resorption per litter with resorptions

1.1 ± 0b

1.7 ± 1.1

1.2 ± 0.4

1.1 ± 0.4

Body weight gain of dams (g)

102.1 ± 13.5b

104.7 + 17.6

101.1 ± 11.8

 86.8 ±20.5d

Fetal crown-rump length (cm)

4.2 ± 0.1c

4.1 ±0.1

 4.1 ± 0.08

3.8 ±0.2d

Fetal body weight (g)

3.4 ± 0 .2c

3.3 ± 0.3

 3.4 ± 0.2

3.4 ± 0.2d

Weight of placenta (g)

0.48 ± 0.4c

 0.58 + 0.18d

0.53 ±0.04d

 0.51 ±0.0 4 d

a — Percentage of pregnant females given in parentheses,

b — Mean + SD.

c — Mean of litter means + SD.

d — Significantly different (p < 0.05) from control group.

Conclusions:
Based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.
Executive summary:

A study was performed to determine the effects of maternal exposure to the test chemical during prenatal development phase. Female rats were given by gavage every other day from days 8 —20 of gestation an aqueous solution of test chemical at daily doses equal to 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(5800 mg/kg). Females were mated overnight with 17-week-old male rats of the same strain. The morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. The control animals were given, by gavage, an equivalent volume of distilled water. The maternal weight gain, daily food and water consumption intake were monitored throughout the gestation period. The female rats were sacrificed on day 21 of gestation under ethyl ether anesthesia. The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded. A site was judged as a late resorption when macroscopic discrimination between fetal residues and placental material was possible. When this discrimination could not be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each female. Live fetuses were measured for body weight, crown-rump length and examined for external malformations. Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination. The test chemical administration was not associated with increased embryo or fetus intrauterine death rates or congenital defects at any dose level. The mid (0.58 g/kg) and high dose (1.15 g/kg) were reported to be associated with dose-related delays in fetal development and the high dose showed clear maternal toxicity. The test chemical does not cause impairment of physical development or behavioral disturbances. Therefore, based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
580 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
The data is from a Klimisch 2 datasource and therefore provides a robust study summary.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
2 500 mg/m³
Study duration:
chronic
Species:
rat
Quality of whole database:
The data is from a Klimisch 2 datasource and provides a robust study summary.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Reproductive toxicity study:

Various studies have been reviewed to assess the potential of the test chemical to cause the reproductive toxicity. These results include in vivo experimental data for the test chemical. The studies are mentioned below:

Study 1:

A study was performed to determine the effects of maternal exposure to the test chemical during prenatal development phase. Female rats were given by gavage every other day from days 8 —20 of gestation an aqueous solution of test chemical at daily doses equal to 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(5800 mg/kg). Females were mated overnight with 17-week-old male rats of the same strain. The morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. The control animals were given, by gavage, an equivalent volume of distilled water. The maternal weight gain, daily food and water consumption intake were monitored throughout the gestation period. The female rats were sacrificed on day 21 of gestation under ethyl ether anesthesia. The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded. A site was judged as a late resorption when macroscopic discrimination between fetal residues and placental material was possible. When this discrimination could not be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each female. Live fetuses were measured for body weight, crown-rump length and examined for external malformations. Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination. The test chemical administration was not associated with increased embryo or fetus intrauterine death rates or congenital defects at any dose level. The mid (0.58 g/kg) and high dose (1.15 g/kg) were reported to be associated with dose-related delays in fetal development and the high dose showed clear maternal toxicity. The test chemical does not cause impairment of physical development or behavioral disturbances. Therefore, based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.

Study 2:

An other study was performed to determine the effects of maternal exposure to the test chemical during post-natal development phase. 36 nulliparous, non-pregnant rats (10 control; 14 dose group 1, 12 dose group 2) were used for the study. Females were mated overnight with 17-week-old male rats of the same strain. The morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. The inseminated females were divided into one control and 2 treatment groups receiving daily 0.58 and 1.15 g/kg aqueous solutions of the test chemical from day 2 to 20 of gestation which covers complete stages of fetal development, including organogenesis which is considered as a critical stage. Concentrations of test chemical in aqueous solution were adjusted such that the animals received 1 cm3 of solution per 100 g of body weight. The female rats were allowed to deliver and nurse their progeny until they were weaned at 28 days of age. Evaluation of female rat fecundity in terms of the number of live fetuses in a litter was done. The assessment of physical development, viability and behavioral tests were performed. Performance of all pups was assessed in the following tests: surface righting at the age of 3 —6 days; negative geotaxis at the age of 6 —12 days; forepaw suspension at 6 —16 days; and air righting at 14—19 days of life. In all pups age at appearance of eye opening, incisor eruption and pinna unfolding was determined. Randomly chosen offspring from each litter were assessed for exploratory motor activity at the age 8, 11 and 14 weeks, and avoidance acquisition of the adult offspring at age ca. 14 weeks. Exposure of the pregnant females to test chemical at dose 1.15 g/kg decreased hemoglobin level in 5-week old female offspring and at a dose of 0.58 g/kg in young males but did not change hematocrit value in 5- and 8-week old male or female offspring. Observations of the physical development of the offspring revealed that, after reaching sexual maturity (14th-15th week), the male offspring of the female rats receiving the highest dose of test chemical had significantly lower body weight gain than the control male offspring of the same age (57.6 ± 18.4 g vs. 48.2 + 15.1 g). Maternal exposure to test chemical at doses 0.58 and 1.15 g/kg neither changes the age of pups at the time of pinna unfolding, incisor eruption, and eye opening, nor altered the performance of righting reflex, negative geotaxis and forepaw suspension. At the dose of 1.15 g/kg, the chemical significantly increased exploratory motor activity of the female offspring at the age of 8 weeks (to 144% of the respective value for the control group). Prenatal exposure to test chemical at dose 0.58 g/kg and 1.15 g/kg did not affect significantly the locomotor activity of males and the active avoidance acquisition of the adult offspring. Therefore, based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.

Study 3:

A study was performed where the toxic effects of the test chemical on prenatal development were observed and recorded according to a procedure prescribed by OECD TG 414 and EPA TSCA TG 798.4900. The test chemical was administered orally via gavage once daily to female rats on days 6 through 15 of presumed gestation. Dosages of 0 (Vehicle), 125, 250, 500 and 1000 mg/kg/day were administered to the rats using corn oil as the vehicle. The dosages were prepared at concentrations of 0 (Vehicle), 25, 50, 100 and 200 mg/mL, respectively. The dosage volume was 5 mL/kg, adjusted daily on the basis of the individual body weights recorded immediately prior to the administration. Each dosage group consisted of 25 presumed pregnant rats. The rats were examined daily during the dosage and post dosage periods for clinical observations of test chemical effects, abortions, premature deliveries and deaths. Body weights and feed consumption values for the rats were recorded on day 0 of presumed gestation and daily during the dosage and post-dosage periods. On day 20 of presumed gestation, the rats were sacrificed by carbon dioxide asphyxiation, and the abdomen of each rat was opened and examined for pregnancy, number and placement of implantation sites, early and late resorptions, live and dead fetuses and number of corpora lutea. Increased incidences of tail and heart malformations (ventricular septal defects) and a large number of different vertebral variations were observed in the foetuses at 1000 mg/kg body weight and day. At concentrations of 500 mg/kg/day and above, maternal toxicity, reduced body weight gains and reduced feed consumption were recorded at the beginning of the treatment. However, the body weight gains in the dams were reduced slightly (14%) during gestation at 1000 mg/kg body weight and day. The fetal effects observed at 500 mg/kg bw were assessed and it was concluded that the effects observed was not due administration of the test chemical and were considered secondary to reduction in maternal body weight and feed consumption. Therefore, based on all the observations and results, it was concluded that the NOAEL (no observed adverse effect levels) for developmental and maternal toxicity were 500 and 250 mg/kg/ day, respectively.

Study 4:

A study aimed at evaluation of the effects of the test chemical on the fertility and frequency of dominant lethal mutations in germ cells of male rats. The study was performed according to OECD 478 Guidelines, but with slight deviations. The test chemical was administered to 10 Wistar male rats/ group doses of 0.1 LD50 or 0.2 LD50 i,e 0,580 and 1160 mg/kg/day once a day, 5 days a week, throughout 8 weeks via gavage once a day, 5 days a week, throughout 8 weeks. The treated rats were observed for mortality, general appearance, body weight, weight gain. Absolute and relative weights of liver, kidney and spermatic vesicle were noted. Testes, seminical vesicles were weighed and observed for histopathological changes. Behavior and appearance did not differ to any significant degree from controls. One animal died during fifth week in 580 mg/kg/day group, whereas one animal died during third week in the 1160 mg/kg/day group. These mortalities were not associated with the administration of the test chemical. Body Weight gain (percent control gain) was as follows: 580 mg/kg/day - 47%; 1160 mg/kg/day - 52%, which was not considered as an adverse effects of the test chemical. Testis was considered target by histopathology but no changes were observed in fertility. Necrosis of the seminiferous tubules, microscopic epithelium was reported in 1/10 controls, and number of unrelated findings in the 580 mg/kg group and high dose males were observed. Therefore, based on all the observations and results, the NOAEL for the test chemical in male Wistar rats was likely to be considered as 580 mg/kg/day.

Study 5:

A study was performed for evaluation of the effects of the test chemical on the fertility and frequency of dominant lethal mutations in germ cells of male rats through the inhalation route. 14 male Wistar rats were exposed to 2500 mg/m3 of the test chemical through whole body inhalation five days a week, five hours a day for 12 months. The mating ratio was 1:2 (1 male per 2 females) with mating interval once at end of study for one week Concentration of test chemical measured by chromatography using a published procedure. Number of Live pups, Number of Dead pups, Corpora lutea, Number of implantations, pre-implantation loss, number of dead implants were calculated per female. No mortality was observed at any time point in both the dose groups. Necropsy effects were not clearly reported, but it was stated that for the “rats exposed to the test chemical the frequency of necrosis of the spermicidal epithelium was much higher than for the control group rats; but the incidence or other details was not given. Leydig cell lesions were also mentioned but it was also stated that the treated and control groups were not different regarding this lesion. Neither implants per female, live fetuses per female nor preimplantation loss per female was affected by treatment. Therefore, based on all the available data, it was concluded that the NOAEL of the test chemical for maternal toxicity is likely to be considered to as 2500 mg/m3 in rats.

Study 6:

The above results are well supported by a one-generation reproduction study performed to determine the toxicity effects of the test chemical. The study was performed according to OECD 415 Guidelines. 5 male Charles River strain rats/dose group and 10 female Charles River strain rats/dose group were used for the study. The test chemical administered of each animal at levels using water as a vehicle at 0.5 or 1.0 percent i.e T-I (Test Dose I): 500mg/kg/day and T-II (Test Dose II): 1000 mg/kg/day). Weighed amounts of the test chemical were blended with tap water to prepare a solution for each test group. Each test solution was thoroughly mixed prior to being divided into the water bottles. Dosing started 90 days prior to mating for males. Mating trials were initiated after males had dosed for 90 days with the test chemical. Mating continued for 15 days or until conception was confirmed. Females were dosed starting with the mating period and throughout gestation, lactation and a 10-day rest period after weaning of F1 litter. Mean female body weight data was collected during the gestation and postpartum periods. Litters of more than 10 pups were reduced to that number by random sacrifice on the fourth day of the lactation period. All pups were examined for physical abnormalities, the number of viable and stillborn members were recorded for all the dose groups. Records of survival were maintained till lactation period and final examination of the physical abnormalities were done on the day of weaning. One T-II male died during the last week of the 90-day treatment period. Necropsy examination revealed the cause of death to be related to respiratory difficulties unrelated to exposure to the test chemical. Significantly lower mean body weights were noted at postpartum days 1 and 4 for T-II (1%) group females. Reduced number of litters were delivered by T-II females. An increased number of stillborn pups were observed in both the test groups. No statistically significant differences were noted between test and control progeny mean body weight values. However, T-I (0.5%) progeny had reduced body weights at the intervals examined when compared to controls. Survival data revealed reduced survival of test progeny than control progeny. Especially noteworthy was the 24 hour survival index of 0 noted in T-II group. Reduced survival rates were noted for T-I group pups at each dose interval examined. Therefore, the Lowest Observed Adverse Effect Level for maternal and F1 generation can be considered to be 500 mg/kg/day when rats were dosed orally with the test chemical mixed in drinking water.

Study 7:

A one generation reproductive toxicity study (according to OECD 415) of the test chemical was performed via inhalation route to assess the effect of the test chemical. Male Charles River COBS albino rats (5/group) were exposed by inhalation to the test chemical at nominal (measured concentrations were close to nominal values) concentrations of 0 or 125 ppm for seven hours a day, five days a week, for 90 days prior to mating with untreated females (10/group, 2/male). The 5 male rats were exposed for the initial 90 days of the chronic inhalation study and then mated with 10 non-treated females for 15 days. Both sexes received exposure mating. Females continued to receive exposure 1 to 2 days prior to delivery. An equal number of untreated control rats were also mated in the same manner. All pups were examined for physical abnormalities, the number of viable and stillborn members were recorded for all the dose groups. Records of survival were maintained till lactation period and final examination of the physical abnormalities were done on the day of weaning. Only a small significant change in the lung/body weight ratio was observed in test group but absence of any histopathological findings indicated that it was due to test chemical exposure. Males used to produce the F1a litter showed no difference in absolute or relative organ weights for brain, gonads, heart, kidneys, liver, or spleen. Absolute lung weighs were reduced by 8 percent in treated males and the relative lung weighs were reduced by 11 percent (statistically significant p<.05). No gross tissue changes which could be attributed to the test chemical were observed. No treatment related effects were noted in F1 generation with regard to mean body weights. Treatment did not significantly affect any measured reproductive parameter. There was a tendency toward an overall reduction in the number of pups born and weaned in both the F1a and F1b litters of the dosed group but this was not statistically significant. Therefore, NOAEC value for the parents and F1 progeny can be considered to be 125 ppm.

Based on the available results, the NOAEL's of the test chemical in male and female animals and also the pups was 500 mg/kg/day which is very high compared to the expected exposure levels. Also, the test chemical was comparatively non-toxic to maternal and offspring individuals when administered via inhalation route. Thus, based upon all the above data and considerations the test chemical is not likely to be classified under the category “Reproductive and Developmental’ toxicant as per CLP criteria of classification and labelling.

Effects on developmental toxicity

Description of key information

Developmental Toxicity Study:

Based on all the available observations and results, it was concluded that the test chemical does not cause any defects in any the development and growth of the fetuses even when it is exposed at high dose levels and therefore is not likely to be classified as a 'developmental toxicant or a teratogen' as per the CLP criteria of classification and labelling.

Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Data is from publication
Qualifier:
according to guideline
Guideline:
other: As mentioned below
Principles of method if other than guideline:
To determine the developmental toxicity of test chemical in rats.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
other: Imp:DAK
Details on test animals or test system and environmental conditions:
Test animals:
- Source: breeding colony from the Nofer Institute (Lodz, Poland)
- Age: approximately 14 weeks
- Weight: 199 ± 12 g
- Fasting period before study: not specified
- Housing: not specified
- Diet: ad libitum, commercial pelleted chow (Fodder Factory, Motycz, Poland)
- Water: ad libitum
- Acclimation period: not specified
Environmental conditions:
- Temperature: approximately 22 °C
- Relative humidity was targeted at 45 – 55 %
- Air changes (per hr): not specified
- Photoperiod: light on between 6 am and 6 pm
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: the inseminated females were divided into one control and three treatment groups receiving 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier found to be 5.8 g/kg.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:Females were mated overnight with 17-week-old male rats of the same strain.
- M/F ratio per cage:
- Length of cohabitation: overnight
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 1 of pregnancy
Duration of treatment / exposure:
From days 8 to 20 of gestation
Frequency of treatment:
Every other day
Duration of test:
Day 21 of gestation
Dose / conc.:
140 mg/kg bw/day
Remarks:
0.025 LD50
Dose / conc.:
580 mg/kg bw/day
Remarks:
0.1 LD50
Dose / conc.:
1 150 mg/kg bw/day
Remarks:
0.2 LD50
No. of animals per sex per dose:
groups of 17-19 pregnant females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the inseminated females were divided into one control and three treatment groups receiving 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(580 mg/kg).
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes / No / No data: YES
- Time schedule: DAILY
- Cage side observations checked in table [No.?] were included. : General appearance of the dosed rats was observed daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / No data: yes
- Time schedule: daily

BODY WEIGHT: Yes / No / No data: yes
- Time schedule for examinations: The maternal weight gain was observed throughout the gestation period

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes / No / No data: The daily food and water
consumption intake were monitored throughout the gestation period.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data: no data available

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes / No / No data: yes
- Time schedule for examinations:The daily water consumption intake were monitored throughout the gestation period.
Ovaries and uterine content:
The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded.
Fetal examinations:
Live fetuses were measured for body weight, crown-rump length and examined for external malformations. Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination.
Statistics:
-In the case of homogeneity of variance, one-way analysis of variance and Dunnett’s test were used: in the case of heterogeneity the Kruskal-Wallis analysis of variance was followed by non-parametric tests.
-Frequency data were analyzed with Fisher’s exact probability test.
-The effect of dioxolane on behavioral performance, food and water consumption and body weight gain of the offspring was evaluated with two-way analysis of variance and Scheffe’s test for multiple comparison.
Indices:
Not specified
Historical control data:
Not specified
Clinical signs:
no effects observed
Description (incidence and severity):
The general appearance and behaviour of the exposed and control animals did not differ significantly
Dermal irritation (if dermal study):
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The toxic effect of the test chemical on the organism of the pregnant female rats was manifested by significantly smaller body weight gain during their pregnancy
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The daily intake of the food of the exposed and control animals did not differ significantly.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
The daily intake of the water of the exposed and control animals did not differ significantly.
Ophthalmological findings:
not specified
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The general appearance and behaviour of the exposed and control animals did not differ significantly.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The toxic effect of test chemical on the organism of the pregnant female rats was manifested by a significantly greater relative mass of the adrenals in the animals receiving the chemical at the highest dose, i.e. 1.15 g/kg. The weight of the placenta in the female rats poisoned with 0.14 g/kg - 1.15 g/kg of this chemical was significantly higher than that in the controls. The dose-effect relationship, however, was not detected.
Gross pathological findings:
not specified
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
not specified
Histopathological findings: neoplastic:
not specified
Other effects:
not specified
Details on results:
not specified
Number of abortions:
not specified
Pre- and post-implantation loss:
not specified
Total litter losses by resorption:
not specified
Early or late resorptions:
not specified
Dead fetuses:
not specified
Changes in pregnancy duration:
not specified
Changes in number of pregnant:
not specified
Other effects:
not specified
Details on maternal toxic effects:
not specified
Dose descriptor:
NOAEL
Effect level:
580 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Fetal body weight changes:
effects observed, treatment-related
Description (incidence and severity):
Teat chemical administered to the maternal rats at the dose of 1.15 g/kg (0.2 LD50) showed fetotoxic effect, causing a reduction of fetus body weights and lengths.
Reduction in number of live offspring:
not specified
Changes in sex ratio:
not specified
Changes in litter size and weights:
effects observed, treatment-related
Description (incidence and severity):
Teat chemical administered to the maternal rats at the dose of 1.15 g/kg (0.2 LD50) showed fetotoxic effect, causing a reduction of fetus body weights and lengths.
Changes in postnatal survival:
not specified
External malformations:
not specified
Skeletal malformations:
effects observed, treatment-related
Description (incidence and severity):
The frequency of occurrence of the delays in epicranial bones and sternum ossification was significantly higher in the groups which were prenatally exposed to higher doses of the compound.
Visceral malformations:
not specified
Other effects:
no effects observed
Description (incidence and severity):
Macro evaluation of the internal organs of the fetuses did not reveal any macro pathology within the organs in any of the evaluated groups of the animals.
Dose descriptor:
NOAEL
Effect level:
580 mg/kg bw/day
Based on:
test mat.
Sex:
not specified
Basis for effect level:
changes in litter size and weights
Remarks on result:
not determinable due to absence of adverse toxic effects
Abnormalities:
not specified
Developmental effects observed:
no
Lowest effective dose / conc.:
1 150 mg/kg bw/day
Treatment related:
yes

Table 1. Absolute and relative organ weights of female rats receiving daily by gevage test chemical on days 8—20 of gestation.

 

Control

0.14 g/kg

0.58 g/kg

1.15 g/kg

Liver

14

18

18

17

(g)

11.69 ± 1.5“

 11.68 ± 1.27

 11.62 + 1.08

 11.21 + 1.02

(g%)

3.91 + 0.24

 3.86 ± 0.24

3.87 + 0.24

3.85 + 0.23

Kidneys

14

18

18

17

(g)

1.36 + 0.15

1.40 ±0.11

1.37 ± 0.13

1.37 ± 0.12

(g%)

0.46 ± 0.03

0.47 + 0.04

0.46 ± 0.03

0.47 ± 0.04

Adrenals

14

18

18

17

(mg)

71.4 + 6.3

72.9 + 9.0

 74.4 ± 8.2

80.1 ± 13.8

(mg%)

24.2 + 2.6

 24.3 ± 3.8

24.9 ± 2.7

27.7 ± 5.9*

 

Table 2: Effect of test chemical administered within 8 — 20 days of gestation on pregnancy and development in rats

 

Control

0.14 g/kg

0.58 g/kg

1.15 g/kg

Females inseminated

17 (82.4)a

 19 (94.7)

18 (100)

19 (89.5)

Live fetuses per litter

11.8 ± 2.2b

11.3 ± 2.2

11.1 ± 1.6

12.0 ± 2.0

Dead fetuses per litter

0

0

0.06 ± 0.24

0.12 ± 0.33

Litters with resorptions

3

10

9

7

Litters with late resorptions

2

6

4

4

Resorption per litter with resorptions

1.1 ± 0b

1.7 ± 1.1

1.2 ± 0.4

1.1 ± 0.4

Body weight gain of dams (g)

102.1 ± 13.5b

104.7 + 17.6

101.1 ± 11.8

 86.8 ±20.5d

Fetal crown-rump length (cm)

4.2 ± 0.1c

4.1 ±0.1

 4.1 ± 0.08

3.8 ±0.2d

Fetal body weight (g)

3.4 ± 0 .2c

3.3 ± 0.3

 3.4 ± 0.2

3.4 ± 0.2d

Weight of placenta (g)

0.48 ± 0.4c

 0.58 + 0.18d

0.53 ±0.04d

 0.51 ±0.0 4d

a — Percentage of pregnant females given in parentheses,

b — Mean + SD.

c — Mean of litter means + SD.

d — Significantly different (p < 0.05) from control group.

Table 3: Effect of test chemical in the incidence of fetuses retarded in development

 

Control

140 mg/kg

580 mg/kg

1150 mg/kg

No. litters examined

14

18

18

17

No. fetuses external exam

157

203

199

204

No. fetuses visceral exam

80

99

98

101

No. fetuses skeletal exam

77

104

101

103

Findings : number

(% incidence of finding)

 

 

 

 

Incomplete development

of skull bones

1 (1.3)

8 (7.7)

12 (12.1)*

52 (50.5)*

Absence of one or several

sternum ossifications

1 (1.3)

7 (6.7)

3 (3.0)

13 (12.6)*

Shortening of 13th ribs

0

0

3 (3.0)

1 (1.0)

* — Significantly different (p < 0.05) from the control group.

Conclusions:
Based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.
Executive summary:

A study was performed to determine the effects of maternal exposure to the test chemical during prenatal development phase. Female rats were given by gavage every other day from days 8 —20 of gestation an aqueous solution of test chemical at daily doses equal to 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(5800 mg/kg). Females were mated overnight with 17-week-old male rats of the same strain. The morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. The control animals were given, by gavage, an equivalent volume of distilled water. The maternal weight gain, daily food and water consumption intake were monitored throughout the gestation period. The female rats were sacrificed on day 21 of gestation under ethyl ether anesthesia. The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded. A site was judged as a late resorption when macroscopic discrimination between fetal residues and placental material was possible. When this discrimination could not be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each female. Live fetuses were measured for body weight, crown-rump length and examined for external malformations. Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination. The test chemical administration was not associated with increased embryo or fetus intrauterine death rates or congenital defects at any dose level. The mid (0.58 g/kg) and high dose (1.15 g/kg) were reported to be associated with dose-related delays in fetal development and the high dose showed clear maternal toxicity. The test chemical does not cause impairment of physical development or behavioral disturbances. Therefore, based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
580 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Data is from a Klimisch 2 datasource and provides a robust study summary.
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

Developmental toxicity study:

Various studies have been reviewed to assess the developmental toxicity potential of the test chemical. These results include in vivo experimental data for the test chemical. The studies are mentioned below:

Study 1:

A study was performed to determine the effects of maternal exposure to the test chemical during prenatal development phase. Female rats were given by gavage every other day from days 8 —20 of gestation an aqueous solution of test chemical at daily doses equal to 0.025, 0.1 and 0.2 of median lethal dose of the test chemical which had been earlier observed to be 5.8 g/kg(5800 mg/kg). Females were mated overnight with 17-week-old male rats of the same strain. The morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. The control animals were given, by gavage, an equivalent volume of distilled water. The maternal weight gain, daily food and water consumption intake were monitored throughout the gestation period. The female rats were sacrificed on day 21 of gestation under ethyl ether anesthesia. The uterus was opened and the number of live fetuses, dead fetuses, early and late resorption sites were recorded. A site was judged as a late resorption when macroscopic discrimination between fetal residues and placental material was possible. When this discrimination could not be made, the site was judged as an early resorption. Total implantation was calculated as the sum of the number of fetuses and resorption sites in each female. Live fetuses were measured for body weight, crown-rump length and examined for external malformations. Approximately half of the live fetuses from each litter were preserved in 95% ethanol for subsequent skeletal examination after staining with Alizarin S. The remaining fetuses were fixed in Bouin’s fluid for visceral examination. The test chemical administration was not associated with increased embryo or fetus intrauterine death rates or congenital defects at any dose level. The mid (0.58 g/kg) and high dose (1.15 g/kg) were reported to be associated with dose-related delays in fetal development and the high dose showed clear maternal toxicity. The test chemical does not cause impairment of physical development or behavioral disturbances. Therefore, based on all the available data and under the given study conditions and observations, the developmental and maternal NOAEL were both judged to be 580 mg/kg/day.

Study 2:

A developmental toxicity study was performed to determine the toxic effects of the test chemical during prenatal development phase. Female rats were administered aqueous doses equal to (0.025, 0.1 and 0.2 LD 50) 0, 580 and 1150 mg/kg bw via oral route. Females were mated overnight with 17-week-old male rats of the same strain, the morning when copulation plugs or spermatozoa appeared in vaginal smears was regarded as day 1 of gestation. Performance of all pups was assessed in the following tests: surface righting at the age of 3 —6 days; negative geotaxis at the age of 6 —12 days; forepaw suspension at 6 —16 days; and air righting at 14—19 days of life. Exposure of the pregnant females to test chemical at dose 1150 mg/kg decreased hemoglobin level in 5-week old female offspring and at a dose of 580 m/kg in young males but did not change hematocrit value in 5- and 8-week old male or female offspring. Maternal exposure to test chemical at doses 580 mg/kg and 1150 mg/kg neither changes the age of pups at the time of pinna unfolding, incisor eruption, and eye opening, nor altered the performance of righting reflex, negative geotaxis and forepaw suspension. At the dose of 1150 mg/kg, the chemical significantly increased exploratory motor activity of the female offspring at the age of 8 weeks (to 144% of the respective value for the control group). Prenatal exposure to test chemical at dose 580 mg/kg and 1150 mg/kg did not affect significantly the locomotor activity of males and the active avoidance acquisition of the adult offspring. Observations of the physical development of the offspring revealed that, after reaching sexual maturity (14th-15th week), the male offspring of the female rats receiving the highest dose of test chemical had significantly lower body weight gain than the control male offspring of the same age (57.6 ± 18.4 g vs. 48.2 + 15.1 g). The viability of the offspring of the female rats receiving test chemical at the highest dose (1150 mg/kg) was significantly lower than that for the control group, and the death rate among the young animals was significantly higher than in the controls. Based on all the observations and results, it was concluded that the test chemical did not show any adverse effect on maternal and fetal parameters and therefore the NOAEL was considered to be 580 mg/kg bw.

Study 3:

A developmental toxicity study was performed in rats to assess the potential developmental toxicity and teratogenicity of the test chemical. Charles River Crl:CDBR VAF/Plus rats were administered a test concentration of 0, 125, 250, 500 and 1000 mg/kg bw via oral gavage route in corn oil. The test chemical was prepared weekly at four concentrations (prepared twice overall) was analyzed for content of test chemical and each batch was analyzed seven days later to establish stability. Females were approximately 7 weeks old at first dosing and Males were approximately 24 weeks old when bred to the virgin females. 160 healthy virgin female rats were placed in cohabitation with 160 breeder male rats in 1:1 ratio (one male rat per female rat). The remaining 20 virgin female rats were placed in cohabitation with breeder male rats that had mated during the first night of the cohabitation period, vaginal lavage or a copulatory plug referred to as day 0 of pregnancy. After completinng the dosing schedule, the intact gravid uterus was excised and weighed. The number and placement of implantation sites, early and late resorptions, live and dead fetuses, and the number of corpora leutea in each ovary were recorded. Uteri from rats that appeared non pregnant were stained with ammonium sulfide to confirm pregnancy status. Litter size, placental weight, gross malformations, fetal body weight, sex ratio, body cross sections, and skeletal examination. Live fetuses were sacrificed by immersion in the appropriate fixative. Approximately one-half of the fetuses in each litter were examined for soft tissue alterations by using a variation of Wilson's sectioning technique. The remaining fetuses in each litter were cleared, stained with alizarin red S(7) and examined for skeletal alterations. Based on the results of this study, the test chemical is not considered a specific developmental toxin. The developmental NOEL was found to be 500 mg/kg/day while the maternal NOEL was found to be 250 mg/kg/day. Neither deaths nor adverse clinical signs were observed in any of the animals. No adverse effects are anticipated for the conceptus in the absence of maternal toxicity. The maternally-toxic 1000 mg/kg/day dosage of the test substance significantly reduced (p<0.05 to p<0.01) fetal body weights for male and female fetuses and for the combined sexes. This dosage of the test substance also significantly increased (P<0.05 to P<0.01) the litter and fetal incidences of externally evident tail malformations, vertebral malformations interrelated with the tail malformations and septal defects in the heart, and significantly reduced (P<0.0l) the litter average for the average number of ossified metacarpal bones per fetus, an expected delay in ossification related to the significantly decreased (P<0.01) fetal body weights. Thus, based on all the observations and results, it was concluded that the NOAEL of the test chemical for both maternal and fetal parameters were considered to be 500 mg/kg bw.

Study 4:

A one generation reproductive toxicity study was conducted in order to ascertain the potential effects, if any, after the oral administration of test chemical upon the fertility and offsprings of albino rats. Charles River strain albino rats were dosed at a concentration of 0.5% and 1.0% (250 mg/kg bw and 500 mg/kg bw) respectively via oral route in drinking water. The test chemical was prepared weekly, and added to the water bottles. The water bottles were changed weekly. Because the water bottles were changed only weekly, the volatility of the test material was determined to not have impacted the study results. The test chemical was available seven days a week, 24 hours per day. Dosing started 90 days prior to mating for males. Females were dosed starting with the mating period and throughout gestation, lactation and a 10-day rest period after weaning of F1 a litter before mating a second time. Following weaning of the F1b litter, all surviving female rats were necropsied. Early deaths were also necropsied. Weights of adrenal glands, gonads, pituitary glands and uterus were recorded at necropsy. Fixed tissues (adrenals, ovaries, pituitary glands, uterus and all apparent lesions) were sectioned and stained with Hemotoxylin & Eosin for microscopic examination. Drinking water exposure of rats at 0.5 or 1.0% produced clear adverse effects on reproduction. In the F1a mating, it could not be determined if effects were attributable primarily to exposure of males, females or both. Survival of high dose pups was especially affected. In the second exposure using untreated proven-male breeders, the effects were less severe suggesting that there was a male contribution to the reproductive toxicity. This conclusion is clouded, however, since the females were not exposed to test chemical during mating, gestation or lactation during the production of this second litter. It is clear that the females were still affected and therefore the female was an affected sex regarding reproductive toxicity of test chemical. Thus from the above observations the LOAEL for maternal and developmental toxicity was considered to be 0.5% (250 mg/kg bw).

Study 5:

A one generation reproductive toxicity study (according to OECD 415) of the test chemical was performed via inhalation route to assess the effect of the test chemical. Male Charles River COBS albino rats (5/group) were exposed by inhalation to the test chemical at nominal (measured concentrations were close to nominal values) concentrations of 0 or 125 ppm for seven hours a day, five days a week, for 90 days prior to mating with untreated females (10/group, 2/male). The 5 male rats were exposed for the initial 90 days of the chronic inhalation study and then mated with 10 non-treated females for 15 days. Both sexes received exposure mating. Females continued to receive exposure 1 to 2 days prior to delivery. An equal number of untreated control rats were also mated in the same manner. All pups were examined for physical abnormalities, the number of viable and stillborn members were recorded for all the dose groups. Records of survival were maintained till lactation period and final examination of the physical abnormalities were done on the day of weaning. Only a small significant change in the lung/body weight ratio was observed in test group but absence of any histopathological findings indicated that it was due to test chemical exposure. Males used to produce the F1a litter showed no difference in absolute or relative organ weights for brain, gonads, heart, kidneys, liver, or spleen. Absolute lung weighs were reduced by 8 percent in treated males and the relative lung weighs were reduced by 11 percent (statistically significant p<.05). No gross tissue changes which could be attributed to the test chemical were observed. No treatment related effects were noted in F1 generation with regard to mean body weights. Treatment did not significantly affect any measured reproductive parameter. There was a tendency toward an overall reduction in the number of pups born and weaned in both the F1a and F1b litters of the dosed group but this was not statistically significant. Therefore, NOAEC value for the parents and F1 progeny can be considered to be 125 ppm.

Based on the available results, the NOAEL's of the test chemical in male and female animals and also the pups was 500 mg/kg/day which is very high compared to the expected exposure levels. Also, the test chemical was comparatively non-toxic to maternal and offspring individuals when administered via inhalation route. Thus, based upon all the above data and considerations the test chemical is not likely to be classified under the category “Reproductive and Developmental’ toxicant as per CLP criteria of classification and labelling.

Justification for classification or non-classification

Based on the available results, the NOAEL's of the test chemical in male and female animals and also the pups was 500 mg/kg/day which is very high compared to the expected exposure levels. Also, the test chemical was comparatively non-toxic to maternal and offspring individuals when administered via inhalation route. Thus, based upon all the above data and considerations the test chemical is not likely to be classified under the category “Reproductive and Developmental’ toxicant as per CLP criteria of classification and labelling.

Additional information