cyproconazole (ISO); (2RS,3RS;2RS,3SR)-2-(4-chlorophenyl)-3-cyclopropyl-1-(1H-1,2,4-triazol-1-yl)butan-2-ol

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

- Oral: NOAEL for systemic toxicity = 20 ppm (dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively); NOAEL for reproductive toxicity = 350 ppm (dietary equivalent to 9.6 and 11.6 mg/kg bw/day for males and females, respectively); NOAEL for developmental toxicity = 20 ppm (dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively), OECD TG 416, Eschbach 1987

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb 1986 to 1 Dec 1986

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-4 (Reproduction and Fertility Effects)

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

KFM

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: F0 8 weeks , F1: 15 weeks
- Housing: Individually, (except mating period) in MacrolonR (polycar­ bonate) cages (size 3) equipped with food and water dispensers (according to Swiss law of animal protection)
- Diet: ad libitum (renewed weekly)
- Water: Municipal water in polypropylene bottles, ad libitum renewed weekly.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: 13 Feb 1986 to 1 Dec 1986

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Premix: 50 g of test substance were mixed with 4950 g of rat diet in a TurbulaR radial mixer for 1 hour to produce a Premix with a concentration of 1%. The Premix was prepared and renewed at monthly intervals. It was kept refrigerated.
- Final diets: Were prepared weekly by mixing Premix with additional rat diet (TurbulaR radial mixer for 1 hour), according to the following
specifications/ratio (for one 5 kg diet container).

- Diet A (4 ppm): 2 g Premix+ 4998 grams Fodder
- Diet B (20 ppm): 10 g Premix+ 4990 grams Fodder
- Diet C (120 ppm): 60 g Premix+ 4940 grams Fodder

Control animals received untreated rat diet
From May 30, 1986 and May 27, 1986 respectively the Premix and final diets were prepared in 4000 g units.

Details on mating procedure:

- M/F ratio per cage: 1 male with 1 female
- Length of cohabitation: maximum of 21 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: The female was removed on day 0 and caged individually.
- Females which failed to mate were killed 26 to 28 days after the end of the mating period and examined
- Females which failed to deliver were killed on day 25 post coitum and examined

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance: Samples were analysed at the start of the study and at 6 month intervals, thereafter.
Test substance in diet: Premix and triplicate aliquots of 10 grams of each experimental diet were analysed for the test substance content at the start of treatment and every second month, thereafter.

Duration of treatment / exposure:

F0: 70 days prior to mating and throughout the mating period, males additional 3 weeks after termination of mating period, females throughout gestation, parturition and lactation period.
F1: 84 days prior to mating until necropsy for all animals.

Frequency of treatment:

Continuously

Details on study schedule:

- Selection of parents from F1 generation when pups were weaned
- Age at mating of the mated animals in the study: approximately 15 weeks

Dose / conc.:

4 ppm

Remarks:

Low dose: Equivalent to mean intake of 0.33 and 0.39 mg/kg bw/day for males and females, respectively

Dose / conc.:

20 ppm

Remarks:

Mid dose: Equivalent to mean intake of 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Dose / conc.:

120 ppm

Remarks:

High dose: Equivalent to mean intake of 9.6 and 11.6 mg/kg bw/day for males and females, respectively

No. of animals per sex per dose:

F0: 26
F1: 26

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based upon data from a one-generation reproduction pilot-study

Parental animals: Observations and examinations:

CLINICAL OBSERVATIONS:
- Time schedule: All animals were examined daily for clinical signs of toxicity.

BODY WEIGHT:
- Time schedule for examinations: Individual male bodyweights were recorded at weekly intervals throughout the study. Individual female bodyweights were recorded weekly during the pre­mating period, on days 0, 7, 14 and 20 of pregnancy (p.c.) and on days 0, 7, 14 and 21 postpartum (p.p.).

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, individual food consumption was monitored at the same time as the bodyweight recording except during the mating period, in which male and females had access to the same feeder. During this period food consumption was not measured.

Oestrous cyclicity (parental animals):

During mating, vaginal smears were taken daily until sperm was found in the smear.

Sperm parameters (parental animals):

Parameters examined in F0/F1 male parental generations:
Testis and epididymis were removed and fixed in 4% formalin. Histopathological examination of all control and high dose males and any animal that failed to mate were conducted.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum
- Maximum of 4 pups/sex/litter by random selection; excess pups were killed and subjected to necropsy. Litters of less than 8 pups were not altered

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Litter size, sex distribution, clinical signs, and malformations of pups were recorded.

Postmortem examinations (parental animals):

SACRIFICE
- The animals were killed by CO2 asphyxiation.
- Male animals: All surviving animals were killed at approx. 3 weeks after the end of the mating period
- Maternal animals: The females were killed at the time of the weaning of the F1-generation offspring (day 21 post partum)

GROSS NECROPSY
- Gross necropsy: Vagina, cervix, uterus, ovaries, testes, epididymis, seminal vesicles, prostate, coagulating gland, pituitary gland, liver and any abnormal lesions. After fixation in 4% formalin, these tissues were embedded in paraffin wax.

HISTOPATHOLOGY / ORGAN WEIGHTS
- Histopathological examination of all listed tissues of all control and high dose animals and any animal that failed to mate or failed to deliver were conducted. Additionally, the liver of all P0 low-, and mid dose males were investigated due to findings in the high-dose group.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed after weaning (day 21).
- These animals were subjected to postmortem examinations (macroscopic and/or microscopic examination) as follows:

GROSS NECROPSY
- Gross necropsy consisted of an examination of the major organs. Organs or tissues which showed any severe macroscopic abnormalities were removed and fixed in 4% formalin.

HISTOPATHOLOGY
Microscopic examination was undertaken if considered necessary.

Statistics:

A computer program automatically subjects all measured values to a parametric or nonparametric statistical analysis. The particular analysis chosen depends on the distribution of values.

Reproductive indices:

Copulation rate, pregnancy rate, and precoital interval were calculated.

Offspring viability indices:

In an adendum to the report, pre/perinatal loss mean incidence was calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Patchy fur loss was observerd in all groups. No toxic signs or symptoms were observed in any male or female rat of the low- and mid-dose groups.
One control male showed a localised, minor skin ulceration for a period of 4 weeks during the premating period. One pregnant high dose female, which failed to deliver, showed signs of distress such as increased respiration rate and piloerection. No signs were seen in any other animal.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animal died during the study. There were no unscheduled sacrifice in the control- and low-dose group except the non-pregnant ones. One female of the mid-dose group and another of the high-dose group was killed because of complete postnatal litter loss on day 1 and 5 p.p., respectively. A pregnant high-dose female which failed to deliver was sacrificed on day 25 post coitum.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Slight fatty change was noted at increased incidence in livers of high dose F0 males. No other treatment related changes were observed.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Indices reflecting mating success were comparable between treated groups and controls. Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F0 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. A slightly lower number of implantation sites was found in the high-dose F0 females, but the value remained within the historical range and is therefore considered not treatment related. The mean pregnancy length did not vary statistically significant among groups

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 350 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 9.6 and 11.6 mg/kg bw/day for males and females, respectively.

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Patchy fur loss was observed in all groups. No toxic signs or symptoms were observed in any male or female rat of the low- and mid-dose groups. One male of the low-dose showed blood-coloured encrustation on the lids of the left eye since the premating period. One female of the control group showed a dark left eye since the mating period. No other toxic signs or symptoms were observed in any male or female rat

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

No parental animal died during the study. No female was sacrificed in the control-, low- or mid-dose group except the non-pregnant ones. One female of the high-dose group was sacrificed as a consequence of complete postnatal litter loss on day 4 p.p.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

A number of common microscopic findings were observed in the organs examined. The type, incidence, and severity of these findings were considered not treatment related.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Indices reflecting mating success were comparable between treated groups and controls. Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F1 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. The mean pregnancy length did not vary statistically significant among groups.

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL) Systemic toxicity

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Key result

Dose descriptor:

NOAEL

Remarks:

Toxicity to reproduction

Effect level:

> 350 ppm

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

Dietary equivalent to 9.6 and 11.6 mg/kg bw/day for males and females, respectively.

Key result

Critical effects observed:

no

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

A dose-related increase in pre/perinatal mortality in the mid- and high-dose groups (13.6% and 16.3%, respectively). The pre/perinatal mortality was also considered relatively high in the control group by the author at 10.7%.
There was a corresponding slight increase in postnatal mortality (days 0 – 21 p.p) in the mid- and high-dose groups (6.6% and 8.1%). Post-natal days 0 - 4 were most affected. A further analysis of the data (reported in an addendum to the report) showed that the apparent increase in pre-perinatal and post natal mortality at the mid-dose resulted from one single F0-dam which lost 100% of its pups (12/13 pre/perinatal losses, the single surviving pup dying in the first days). The mode of calculation of the indices gives a strong weight to these losses, and since no confirmation of these effects were found in the F2 litters, they were considered not treatment-related. In the high-dose, increased post-natal losses are also due to one single dam in each generation. Nevertheless, since the loss rats for both the pre/perinatal and the post natal periods are constantly high in both generations, an effect of treatment cannot be excluded.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Hepatic hemorrhage was observed in 2 rats, extramedullary hemopoiesis in the liver of 1 rat, hydronephrosis in 7 rats, and renal hemorrhage in 1 rat. All these findings were considered not treatment related.

Other effects:

not examined

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F1

Effect level:

20 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

In the F2 litters there was a slight increase in pre/perinatal mortality in the high dose only. There was a slightly higher post-natal mortality during days 0-4 in the F2 high dose group only (7.6% greater than controls).

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

not examined

Nipple retention in male pups:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Non treatment related effects were observed: Hepatic congestion in 6 rats, extramedullary hemopoiesis in the liver of 7 rats, hepatocellular fatty change in 3 rats, hydronephrosis in 40 rats, lymphoid hyperplasia in the cervical lymph node of 1 rat, and hyperemia in the cervical lymph node of 2 rats.

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Generation:

F2

Effect level:

20 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Remarks on result:

other:

Remarks:

Dietary equivalent to 1.6 and 1.9 mg/kg bw/day for males and females, respectively

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Table 1. Actual substance intake in mg/kg bw/day

	4 ppm		20 ppm		120 ppm
	Males	Females	Males	Females	Males	Females
F0	0.28	0.33	1.39	1.67	8.29	9.88
F1	0.37	0.45	1.77	2.16	10.88	13.30

Table 2. Liver weights of parental animals

	males				females
	0 ppm	4 ppm	20 ppm	120 ppm	0 ppm	4 ppm	20 ppm	120 ppm
F0
body weight (g)	425	433	425	431	268	265	268	270
liver weight (g)	13.8	14.3	14.0	14.7	14.0	13.4	14.1	14.8
rel. liver weight (% of bw)	3.25	3.31	3.28	3.43*	5.26	5.05	5.25	5.49
F1
body weight (g)	470	459	464	462	273	275	269	271
liver weight (g)	16.0	15.9	16.2	16.3	14.0	14.7	14.3	14.5
rel. liver weight (% of bw)	3.42	3.48	3.50	3.52	5.12	5.35	5.31	5.36

*) P<0.05

 Table 3. Mean reproductive parameters

Parameter	Generation	0 ppm	4 ppm	20 ppm	120 ppm
Females on study	F0	26	26	26	26
	F1	26	26	26	26
Females mated (Mating index (%))	F0	26 (100)	26 (100)	26 (100)	26 (100)
	F1	26 (100)	26 (100)	26 (100)	25 (96.2)
Females pregnant (Fertility index (%))	F0	26 (100)	23 (88.5)	24 (92.3)	25 (96.2)
	F1	22 (84.6)	22 (84.6)	24 (92.3)	22 (88.0)
Females with liveborn (Gestation index (%))	F0	26 (100)	23 (100)	24 (100)	24^a)(96.0)
	F1	22 (100)	22 (100)	24 (100)	22 (100)
Mean duration of gestation (days)	F0	22.3	22.4	22.6 (increase)	22.5 (increase)
	F1	22.3	22.5	22.2	22.3
Mean implantation site per dam	F0	12.5	12.4	12.4	11.6 (decrease)
	F1	12.4	12.4	12.7	12.4

a) One female failed to deliver and was killed

Table 4. 2- generation rat litter data

Parameter	Generation	0 ppm	4 ppm	20 ppm	120 ppm
Mean live pups/dam at days					9.8(-) 7.2(-) 7.1(-) 10.9 (-) 7.2 (-) 7.1 (-)
0 4a)	F1	11.2 7.8	11.5 7.7	10.8 7.5
21		7.8	7.7	7.4
0 4a)	F2	11.0 7.7	11.7 7.7	12.0 7.8
21		7.7	7.6	7.7
Pre- and perinatal loss (%)	F1 F2	10.7 11.3	7.8 7.3	13.6(+) 5.6	16.3(+) 12.6(+)
Mean postnatal loss (%) at days 0 - 4	F1	0.3	1.6	5.6	7.6(+)
0 - 21 0 - 4 0 - 21	F2	0.3 2.2 2.2	1.6 4.2 5.9	6.6 1.4 2.9	8.1(+) 5.8(+) 7.6(+)
Sex ratio at birth (males/females)	F1 F2	0.49 0.48	0.47 0.50	0.49 0.49	0.49 0.55
Mean pup weight at birth (g)
male	F1	5.9	5.8	5.9	5.9
female		5.6	5.5	5.6	5.7
male	F2	5.8	6.0	5.8	6.0
female		5.5	5.8	5.4	5.7
Mean pup weight at day 21 p.p.
male	F1	45.8	45.4	46.4	44.2
female		44.1	44.6	44.4	43.5
male	F2	46.2	49.4	49.0	47.1
female		44.9	47.6	46.9	45.5

a) after culling
(-) Decrease

(+) Increase

Conclusions:

Under the conditions of this study, parental toxicity for liver effects was observed at the highest dose, resulting in a NOAEL of 20 ppm. Based on a dose related increase in pre/perinatal mortality in the high-dose groups in the F0 and F1 generation, 20 ppm is considered to be the NOAEL for reproductive toxicty. This dose corresponds to 1.4 and 1.7 mg/kg bw in F0 males and females and 1.8 and 2.16 mg/kg bw/day in F1 males and females, respectively

Executive summary:

In a 2 -generation reproduction study that was performed according to OECD TG 416 and GLP principles, 26 male and 26 female KFM-Wister rats per group were exposed to the test substance in 4 groups at levels of 0, 4, 20 and 120 ppm in the diet. The test substance was administered to the parental (F0)- animals during premating (10 weeks), mating, the resulting pregnancy and through weaning their offspring. Furthermore randomly selected F1-pups (4 male + 4 female/ litter if possible) were treated in the same way as the F0 animals except the premating period which covered a period of 12 weeks. Parental (F0 and F1) body weight, food consumption, clinical signs and mortality during all stages of the study, liver weight ratios, histopathological examination of reproductive system and liver, copulation- and pregnancy rates, precoital intervals and pregnancy lengths were investigated. Weanlings (F1 and F2) litter data, pre/peri- and postnatal mortality, postnatal bodyweight development and clinical signs (days 0 - 21 postpartum), sex distribution and histopathological examination of findings were investigated.

Results showed that no treatment related clinical signs were noted in either the F-0 or F-1 parental generation and no parental animals died during treatment. The only clinical signs noted in F-0 animals were in a single high dose female that failed to deliver and showed signs of distress and increased. respiration. Clinical signs in the F-1 generation were minor and did not appear to be treatment related. There were no unscheduled deaths in the controls or low dose groups of the F0 or F1. One female of the mid-dose group and one from the F0 high dose groups were sacrificed because of total litter losses on day 1 and 5 post-partum, respectively. In addition, a single F0 high dose female, which failed to give birth, was sacrificed on day 25 post-coitum. A single high dose F1 female was sacrificed following complete postnatal litter loss on day 4 post-partum. Body weight and food consumption were comparable between treated groups and controls during the entire study period. A marginal/slight increase in relative liver weight was seen in F0 males (5.5% greater than controls) and females (4.4% greater than controls), attaining statistical significance in males only (2p > 0.05). Slight fatty change was noted at increased incidence in high dose F0 males. No other treatment related changes were observed. Indices reflecting mating success were comparable between treated groups and controls (F0+F1). Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F0 and F1 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. The mean pregnancy length in the low dose F0 and F1 females was comparable to their respective controls. An increased gestation length was observed in some mid- and high-dose F0 females. A single mid-dose animal delivered a single pup on day 24 of gestation. A single female of the high F0 group failed to deliver and was not included in the calculations of gestation length. However, this female had only a single implantation site and no foetuses at termination. In the F0 generation the females delivered on days 22 or 23, however the distribution between those two days varied among groups. Delivery on day 22 occurred for 66% of the controls and 50% of the dams in the 20 and 120 ppm groups, indicating a slight increase in the length of pregnancy in some animals of these dose groups, which may have been related to treatment. However, there was no such increase in the F1 generation dams. There was a slight reduction in mean implantation sites in the F0 high dose animals (-7%vs. controls). A decreased litter size at birth was seen in this group (-12% than controls). Neither parameter was affected in the F1 generation. Among pups in the F1 generation there was a dose-related increase in pre/perinatal mortality in the mid- and high-dose groups (13.6% and 16.3%,respectively). The per/perinatal mortality was also considered relatively high in the control group by the author at 10.7%. There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the mid- and high-dose groups (6.6%c and 8.1%). Post-natal days 0 – 4 were most affected. However, the apparent increases at 20 ppm in pre/peri and post-natal mortality in the F1 litters result from one single F0-dam which lost 100% of its pups (12/13 pre/perinatal losses, the single surviving pup dying in the first 4 days). As there was no effect in the F1 generation, this event was unlikely to be treatment-related. In the F2 litters there was a slight increase in pre/perinatal mortality in the high dose only. There was a slightly higher post-natal mortality during days 0-4 in the F2 high dose group only (7.6% greater than controls). Pup weights were not affected in either F0 or F1. No treatment-related alterations were identified in pups on post-mortem examination.

In conclusion, administration of the test substance in the diet of rats in this two-generation reproduction study produced minimal signs of parental toxicity in F0 males at the highest dose tested (NOAEL 20 ppm). A slight (statistically significant) increase in relative liver weight was associated with a marginally increased incidence in liver fatty change. This observation is of questionable significance as no toxicity was seen in F0 females or F1 males and females. In addition, the 90-day rat studies indicated that the NOAEL for liver effects was in excess of 80 ppm (6.4 mg/kg bw/day). An increase in gestation in some high dose F0 females may have been related to treatment, but was not observed in the F1generation. In the F0 generation there was a slight, statistically non-significant decrease in the number of implants in the high-dose group. In addition, there was a dose related increase in pre/perinatal mortality in the high-dose groups in the F0 and F1 generation (16.3% and 12.6%, respectively). There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the high-dose group of the F0 and F1 (8.1% and 7.6%, respectively. Treatment with the test substance had no effects at 4 ppm. The dose level of 20 ppm could be regarded as a NOAEL in this study if the increased gestation length and litter loss of the single 20 ppm female are disregarded. This dose corresponds to 1.4 and 1.7 mg/kg bw in F0 males and females and 1.8 and 2.16 mg/kg bw/day in F1 males and females, respectively.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

1.4 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

A guideline study (comparable to OECD 416) performed in compliance with GLP

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Quality of whole database:

A guideline study (comparable to OECD 416) performed in compliance with GLP

Additional information

All available data was assessed. One 2-generation reproduction toxicity study (OECD TG 416), performed under GLP study, was available and included as key study. One developmental toxicity study in rats and one developmental toxicity study in rabbits were included as key studies. One developmental toxicity study in rats from published data was available and included as supporting study. One other developmental toxicity study in rabbits was also available and included as supporting study.

 

Toxicity to reproduction

In a 2-year generation reproduction study that was performed according to OECD TG 416 and GLP principles (Eschbach 1987), 26 male and 26 female KFM-Wister rats per group were exposed to the test substance in 4 groups at levels of 0, 4, 20 and 120 ppm in the diet (dietary equivalent to 0, 0.33, 1.6, 9.6 mg/kg bw/day for males and 0, 0.39, 1.9, 11.6 mg/kg bw/day for females). The test substance was administered to the parental (F0)- animals during premating (10 weeks), mating, the resulting pregnancy and through weaning their offspring. Furthermore randomly selected F1-pups (4 male + 4 female/ litter if possible) were treated in the same way as the F0 animals except the premating period which covered a period of 12 weeks. Parental (F0 and F1) body weight, food consumption, clinical signs and mortality during all stages of the study, liver weight ratios, histopathological examination of reproductive system and liver, copulation- and pregnancy rates, precoital intervals and pregnancy lengths were investigated. Weanlings (F1 and F2) litter data, pre/peri- and postnatal mortality, postnatal bodyweight development and clinical signs (days 0 - 21 postpartum), sex distribution and histopathological examination of findings were investigated.

Results showed that no treatment related clinical signs were noted in either the F-0 or F-1 parental generation and no parental animals died during treatment. The only clinical signs noted in F-0 animals were in a single high dose female that failed to deliver and showed signs of distress and increased. respiration. Clinical signs in the F-1 generation were minor and did not appear to be treatment related. There were no unscheduled deaths in the controls or low dose groups of the F0 or F1. One female of the mid-dose group and one from the F0 high dose groups were sacrificed because of total litter losses on day 1 and 5 post-partum, respectively. In addition, a single F0 high dose female, which failed to give birth, was sacrificed on day 25 post-coitum. A single high dose F1 female was sacrificed following complete postnatal litter loss on day 4 post-partum. Body weight and food consumption were comparable between treated groups and controls during the entire study period. A marginal/slight increase in relative liver weight was seen in F0 males (5.5% greater than controls) and females (4.4% greater than controls), attaining statistical significance in males only (2p > 0.05). Slight fatty change was noted at increased incidence in high dose F0 males. No other treatment related changes were observed. Indices reflecting mating success were comparable between treated groups and controls (F0+F1). Several females in all groups of both generations failed to become pregnant, however, there were no treatment related differences between treated and control groups. The fertility indices in the F0 and F1 females were comparable between treated and control groups. Numbers of implantation sites per dam did not significantly differ among groups. The mean pregnancy length in the low dose F0 and F1 females was comparable to their respective controls. An increased gestation length was observed in some mid- and high-dose F0 females. A single mid-dose animal delivered a single pup on day 24 of gestation. A single female of the high F0 group failed to deliver and was not included in the calculations of gestation length. However, this female had only a single implantation site and no foetuses at termination. In the F0 generation the females delivered on days 22 or 23, however the distribution between those two days varied among groups. Delivery on day 22 occurred for 66% of the controls and 50% of the dams in the 20 and 120 ppm groups, indicating a slight increase in the length of pregnancy in some animals of these dose groups, which may have been related to treatment. However, there was no such increase in the F1 generation dams. There was a slight reduction in mean implantation sites in the F0 high dose animals (-7%vs. controls). A decreased litter size at birth was seen in this group (-12% than controls). Neither parameter was affected in the F1 generation. Among pups in the F1 generation there was a dose-related increase in pre/perinatal mortality in the mid- and high-dose groups (13.6% and 16.3%,respectively). The per/perinatal mortality was also considered relatively high in the control group by the author at 10.7%. There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the mid- and high-dose groups (6.6%c and 8.1%). Post-natal days 0 – 4 were most affected. However, the apparent increases at 20 ppm in pre/peri and post-natal mortality in the F1 litters result from one single F0-dam which lost 100% of its pups (12/13 pre/perinatal losses, the single surviving pup dying in the first 4 days). As there was no effect in the F1 generation, this event was unlikely to be treatment-related. In the F2 litters there was a slight increase in pre/perinatal mortality in the high dose only. There was a slightly higher post-natal mortality during days 0-4 in the F2 high dose group only (7.6% greater than controls). Pup weights were not affected in either F0 or F1. No treatment-related alterations were identified in pups on post-mortem examination.

In conclusion, administration of the test substance in the diet of rats in this two-generation reproduction study produced minimal signs of parental toxicity in F0 males at the highest dose tested (NOAEL 20 ppm). A slight (statistically significant) increase in relative liver weight was associated with a marginally increased incidence in liver fatty change. This observation is of questionable significance as no toxicity was seen in F0 females or F1 males and females. In addition, the 90-day rat studies indicated that the NOAEL for liver effects was in excess of 80 ppm (6.4 mg/kg bw/day). An increase in gestation in some high dose F0 females may have been related to treatment, but was not observed in the F1generation. In the F0 generation there was a slight, statistically non-significant decrease in the number of implants in the high-dose group. In addition, there was a dose related increase in pre/perinatal mortality in the high-dose groups in the F0 and F1 generation (16.3 % and 12.6 %, respectively). There was a corresponding slight increase in postnatal mortality (days 0 - 21 p.p) in the high-dose group of the F0 and F1 (8.1 % and 7.6 %, respectively. Treatment with the test substance had no effects at 4 ppm. The dose level of 20 ppm could be regarded as a NOAEL in this study if the increased gestation length and litter loss of the single 20 ppm female are disregarded. This dose corresponds to 1.4 and 1.7 mg/kg bw in F0 males and females and 1.8 and 2.16 mg/kg bw/day in F1 males and females, respectively.

Effects on developmental toxicity

Description of key information

Oral: NOAEL for maternal toxicity is 10 mg/kg bw/day, the NOAEL for developmental toxicity is 2 mg/kg bw/day, rabbit, equivalent to OECD 414, Müller 1991

Oral: NOAEL for maternal toxicity is 6 mg/kg bw/day; the NOAEL for developmental toxicity is 12 mg/kg bw/day, rat, equivalent to OECD 414, Becker 198

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Jul 1985 to 14 Aug 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1982

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: "Teratogenicity study", Guidelines for Registering Pesticides in the U.S.A., U.S. Environmental Protection Agency, Hazard Evaluation: Human and Domestic Animals. U.S. Federal Reg­ ister, Vol. 43, paragraph 163.83-3, adopted November

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Wistar/HAN rat (Kfm: WIST, outbred, SPF quality)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 11 weeks, minimum
- Weight at study initiation: 180 -236 g
- Housing: The animals were housed individually in Makrolon cages type-3 with wire mesh tops and standardized granulated softwood bedding.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 9 days under test conditions, after veterinary examination

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ±2
- Humidity (%): 55 ±10
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 8 Jul 1985 to 14 Aug 1985

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was weighed into a glass beaker on a tared precision balance and the vehicle added (w/v). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.

DIET PREPARATION
- Rate of preparation of diet (frequency): The test substance/vehicle mixture were prepared daily prior to administration.

VEHICLE
- Concentration in vehicle: 4% CMC
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Determination of concentration, homogeneity and stability of the test article in the vehicle/test article mixtures were performed once during the treatment period. Samples were taken immediately after mixture preparation and again after 90 minutes. Aliquots (about 1 g) were diluted with acetone to give approximate the standard concentrations and injected into the gas chromatograph. Standard solutions in the range of 10 to 100 µg/mL of test substance in acetone were prepared and used for the calibration of the gas chromatograph.

Gas chromatographic conditions:

Apparatus: Packard 428 gas chromatograph with FI-Detector and Shimazu CR3A Integrator
Column: OV-210 10 % on Gaschrom Q 80 - 100 mesh,1.5 m x 2 mm

Temperature: - Injector: 270 degrees centigrade,
- Oven: 230 degrees centigrade,
- Detector: 280 degrees centigrade

Carrier gas, Helium; 30 mL/min.
Injection volume, 3 µL

Details on mating procedure:

- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear or observation of copulation plug referred to as day 0 of pregnancy

Duration of treatment / exposure:

From day 6 through day 15 post coitum

Frequency of treatment:

Daily

Duration of test:

Until day 21 post coitum

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based upon the results of a dose-finding teratogenicity study in rats

Maternal examinations:

CLINICAL OBSERVATIONS:
- Time schedule: Twice daily

BODY WEIGHT:
- Time schedule for examinations: Daily, from day 0 until day 21 post coitum

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes, data were recorded on day 6, 11, 16 and 21 post coitum.

POST-MORTEM EXAMINATIONS:
- Sacrifice on gestation day 21 post coitum
- Organs examined: All internal organs

Ovaries and uterine content:

The ovaries and uterine content was examined after termination:
Examinations included:
- Gravid uterus weight: Yes
- Position of foetuses in the uterus: Yes
- Number of corpora lutea: Yes

OTHER: Caesarean section
- The uteri of all females which were found at necropsy to be not pregnant were placed in an aqueous solution of ammonium sulphide to accentuate possible haemorrhagic areas of implantation sites. All tissues and organs except foetuses removed from animals killed at scheduled necropsy were discarded.

Blood sampling:

No data

Fetal examinations:

- External examinations: Yes
- Skeletal examinations: Yes
- Head examinations: Yes

OTHER: Procedure Wilson technique of the viscera and brain
- Slicing technique of Wilson (1965) for examination of the viscera and brain. One third of the number of live foetuses from each litter was fixed in a mixture of ethyl alcohol, formal and acetic acid. After evaluation the individual sections were preserved in a solution of ethyl alcohol and glycerine (one foetus per container). Descriptions of any abnormalities were recorded.

- The remaining foetuses (two thirds of the number of live foetuses) were placed in a solution of potassium hydroxide for clearing and stained with alizarin red. The skeletons were examined and all abnormalities were recorded. The specimens were preserved individually in plastic bags.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No sign or symptom was observed in any female of the vehicle control group and no test substance related finding was observed in any female of any dose group.

The following common observations were noted:
- Group 2 (6 mg/kg): Two animals, hairless areas on the fur and dullness of both eyes, respectively, were noted on day 6 post coitum.
- Group 3 (12 mg/kg): One animal, hairless areas were noted on day 13 post coitum. In female no. 64, a node (10 x 20 mm) in the left inguinal area was noted on day 19 post coitum
- Group 4 (24 mg/kg) : No sign or symptom was observed.
- Group 5 (48 mg/kg): One female, ruffled fur and whitish flux was noted on day 6 post coitum, and in another animal, hairless areas were noted on day 16 post coitum.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean body weight and mean corrected body weight gain of the 6 and 12 mg/kg bw/day groups were not considered different from controls. There was a reduction in body weights (body weight loss) from day 6-7 and from day 7-8 of treatment in both the 24 and 48 mg/kg bw/day groups. Body weight gain recovered in the following days. There was an overall reduction in body weight gain for the period 6-11. Mean body weight gain was similar to controls for the remaining period at all dose levels. Mean body weights were statistically significant lower (p < 0.05) from day 12 of treatment at ≥ 24 mg/kg bw/day. This was due to the reduction in uterus weight (reduced foetus number) at these dose levels. In an additional evaluation of the body weight gain, a dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

The mean food consumption of groups 4 and 5 was significantly reduced (dose-related) during the treatment period in comparison to that of the vehicle control group. No significant or test substance related differences in food consumption were noted between the vehicle control group and group 2 (6 mg/kg) or 3 (12 mg/kg).

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test substance related finding was noted in any female of any dose group and no abnormal finding was evident in any female of the vehicle control group.

The following incidental common findings were noted:
- Group 3 (12 mg /kg): In one animal, on the left side of the inguinal area, a dark­ red node (10 x 20 mm) was noted between the epidermis and the hypodermis.
- Group 5 (48 mg/kg): In one animal, the uterus was discoloured dark-red and contained reddish turbid fluid. This finding was considered to be the result of the nine dead embryos (5 embryonic and 4 foetal resorptions).

No abnormal finding was noted in groups 2 (6 mg/kg) and 4 (24 mg/kg).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Pre- and post-implantation loss:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (24 mg/kg) and 5 (48 mg/kg), a significantly increased postimplantation loss (dose-related) was noted

Total litter losses by resorption:

effects observed, treatment-related

Description (incidence and severity):

One animal in group 5 (48 mg/kg) had implantation sites only (resorption of all embryos).

Early or late resorptions:

effects observed, treatment-related

Description (incidence and severity):

The total losses consisted mainly of early embryonic resorptions, total resorptions and to a lower extent late resorptions, amounted to 22.2 % in group 4 (24 mg/kg) and 30.6 % in group 5 (48 mg/kg). A single female of the high dose group had resorptions only; inclusion of this female increased the percentage post implantation loss for this group to 34.2%.

Dead fetuses:

effects observed, non-treatment-related

Description (incidence and severity):

2 dead fetuses were found in group 4 (24 mg/kg) and 1 dead fetus in group 5 (48 mg/kg)

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights of groups 1 (vehicle control), 2 (6 mg/kg) and 3 (12 mg/kg) were similar. No test substance related differences were evident.
In groups 4 (24 mg/kg) and 5 (48 mg/kg), the group mean body weights of foetuses were slightly reduced (8.3 %), in comparison with that of the vehicle control group and statistically significant. This finding was considered to be test substance related

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

No significant differences between the sex ratios of foetuses of the dose groups and the vehicle control group were evident.

Changes in litter size and weights:

not examined

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External investigations revealed the presence of one runt in each of the 6, 12, 24 and 48 mg/kg groups. This finding was considered to be incidental and not related to treatment. No other malformed or anomalous foetus was found in the 6, 12, 24 mg/kg groups. At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which one was a runt.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal investigations revealed no treatment related malformations. The frequency of some relatively common findings appeared to be increased in a dose-related manner from 12 mg/kg bw/day, i.e., absent sternebrae and supranummery ribs. It was concluded by the authors that these increases were within the normal range of deviations and variations for animals of this strain. The stages of skeletal ossification in the control, 6 and 12 mg/kg groups were similar and reflected the normal range of variations for animals of this strain and age. In the 24 and 48 mg/kg groups, the incidence of incompletely ossified phalangeal nuclei and calcanea with still absent ossification was increased compared to that of the control. These findings may be the result of the reduced mean bodyweights of foetuses. In addition, the incidence of supernumerary ribs exceeded the historical control range at the two higher dose levels.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Visceral investigations of foetuses by the Wilson technique revealed no abnormal findings in the control, 6 and 12 mg/kg groups. At 24 mg/kg a hydrocephalus internus was noted. At 48 mg/kg the previously mentioned hydrocephalus and the palatoschisis were confirmed. In addition, another hydrocephalus internus was noted in the high dose group.

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

12 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

fetal/pup body weight changes

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: cranium

other: skeletal: incomplete or absence of ossification (phalangeal nuclei and calcanea)

Description (incidence and severity):

At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which
one was a runt. A single incidence of hydrocephalus internus was also apparent at the 24 mg/kg bw/day dose level.
At 24 and 48 mg/kg bw/day incomplete ossification of phalangeal nuclei and
the absence of ossification in calcanea was observed.

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

24 mg/kg bw/day (nominal)

Treatment related:

yes

Relation to maternal toxicity:

developmental effects occurring together with maternal toxicity effects, but not as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Table 1a. Differences in body weight gain of dams over the treatment period

Group (mg/k)	Days post-coitum
	0-6 gms (%)	6 – 11 gms (%)	11 – 16 gms (%)	6 – 16 gms (%)	16 – 21 gms (%)	6 – 21 gms (%)	Corrected body weight gain (%)*
1 (0)	18(+8.7)	17(+7.6)	27(+11.2)	44(+19.9)	48(+17.8)	92(+41)	9.5
2 (6)	18(+8.7)	16(+7.1)	26(+11)	42(+18.7)	51(+19)	93(+41)	8.4
3 (12)	17(+8.1)	14(+6.2)	25(+10.4)	39(+17.3)	53(+20)	92(+40)	7.2
4 (24)	18(+8.8)	12(+5.4)	23(+9.8)	35(+15.7)	43(+17)	78(+35)	9.3
5 (48)	17(+8.2)	11(+4.9)	22(+9.3)	33(+14.7)	42(+16)	75(+33)	9.5

Table 1b Reinterpretation of body weight gain data of dams over days 6 -11 compared to control group

Dose group	Body weight gain (Days 6 to 11)
6 mg/kg/day	-6%
12 mg/kg/day	-18%
24 mg/kg/day	-29%
48 mg/kg/day	-35%

Table 2. Summary of reproductive parameters.

Parameter		0 mg/kg	6 mg/kg	12 mg/kg	24 mg/kg	48 mg/kg
Number of females on study		25	25	25	25	25
Number of pregnant females (%)		24 (96)	22 (88)	23 (92)	25 (100)	23 (92)
Number of non-pregnant females		1	3	2	0	2
Number of females with 100 % intrauterine deaths		1	0	0	0	1
Number of females with live foetuses at necropsy		23	22	23	25	22
Number of corpora lutea: per group mean per dam		286 12.4	277 12.6	296 12.9	299 12.0	281 12.8
Number of implantations: per group mean per dam		261 11.3	259 11.8	279 12.1	279 12.1	255 11.6
Preimplantation loss: per group mean per dam		25 1.1	18 0.8	17 0.7	20 0.8	26 1.2
Live foetuses:	per group	248	248	271	217	177
	mean per dam	10.8	11.3	11.8	8.7*	8.0*
	% of implantations	95.0	95.8	97.1	77.8	69.4
	% males	49.2	46.8	48.3	45.2	48.9
Dead foetuses		0	0	0	2	1
Early resorptions:	per group	12	10	8	56	65
	mean per dam	0.5	0.5	0.3	2.2	3.0
	% of implantations	4.6	3.9	2.9	20.1	25.5
Late resorptions:	per group	1	1	0	4	12
	mean per dam	0.0	0.0	0.0	0.2	0.5
	% of implantations	0.4	0.4	0.0	1.4	4.7
Total resorptions:	per group	13	11	8	60	77
	mean per dam	0.6	0.5	0.3	2.4	3.5
	% of implantations	5.0	4.2	2.9	21.5*	30.2*
Post implantation losses:	per group	13	11	8	60	78
	mean per dam	0.6	0.5	0.3	2.5	3.5
	% of implantations	5.0	4.2	2.9	22.2	30.6
Mean weight of live foetuses:		4.8	4.8	4.7	4.4*	4.4*

*) P ≤ 0.05

Table 3. Summary of foetal findings.

Parameter	0 mg/kg	6 mg/kg	12 mg/kg	24 mg/kg	48 mg/kg
External examinations: Number of foetuses examined No. of malformations (No. litters affected)	248 0 (0)	248 1 (1)	271 1 (1)	217 1 (1)	177 3 (3)
Visceral examinations: Number of foetuses examined No. of malformations (No. litters affected)	78 0 (0)	79 0 (0)	91 0 (0)	71 1 (1)	60 3 (3)
Total number of palatoschisis (litter)					2 (2)
Total number of hydrocephalus (litter)				1 (1)	2 (2)
Skeletal examinations: Number of foetuses examined No. of anomalies (No. litters affected)	170 5 (4)	169 4 (4)	180 10 (7)	146 14 (8)	117 6 (6)
Supernumerary ribs (no.foetuses(%))	7 (2.05)	19(5.6)	31(8.6)	59(20)	65(27.8)

Conclusions:

The maternal NOAEL in this study was 6 mg/kg based on body weight gain reduction during early treatment at 24 mg/kg. The developmental NOAEL was 12 mg/kg based on reduced foetal body weight, increased post implantation loss and increased incidence foetal malformations at 24 and 48 mg/kg.

Executive summary:

A developmental toxicity study in rats was performed in accordance with OECD TG 414 and GLP principles, to determine the potential of the test substance to induce structural and/or other anomalies in the foetus which may arise from the exposure of the mother during pregnancy. The test substance was administered orally by intubation once daily to mated female Wistar rats (outbred, SPF-quality) from day 6 through day 15 post coitum. Animals were treated at dose levels of 0, 6, 12, 24 and 48 mg/kg bw by oral gavage at a constant dosing volume of 10 ml/kg bw. The vehicle that was used in this study was CMC. Each group consisted of 25 mated female rats. On day 21 post coitum, all females were sacrificed and the foetuses removed by caesarean section. The investigations of females/dams and foetuses were performed in accordance with international recommendations. All parameters recorded were evaluated and reported.

Results showed that no death occurred and no substance related signs or symptoms were observed. At necropsy, no test substance related findings were evident in any female of any group. The oral administration of the test substance caused loss of body weight in the dams of group 5 (48 mg/kg), from the first to second and from the second to third day of treatment. Thereafter the body weight gain was similar to that of the vehicle control group. The reason that the body weights remained below that of the vehicle control group was as noted as in group 4 (24 mg/kg), primarily the consequence of the lower number of foetuses per dam. A dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.The mean food consumption of groups 4 and 5 was significantly reduced (dose-related) during the treatment period in comparison to that of the vehicle control group. No significant or test substance related differences in body weight gain and food consumption were noted between the vehicle control group and group 2 (6 mg/kg) or 3 (12 mg/kg). The evaluations of the reproduction data resulted in dose related significantly increased post implantation losses of 22.2 % in group 4 (24 mg/kg) and 34.2 % in group 5 (48 mg/kg). In the vehicle control group and in groups 2 (6 mg/kg) and 3 (12 mg/kg) losses of 5.0 %, 4.2 % and 2.9 % were noted, respectively. In the foetuses, slight but significantly reduced mean body weights of 8.3 % were noted in groups 4 and 5 when compared to that of the vehicle control group. During the external investigations of foetuses, three foetuses of different litters/dams with anomalies were found in group 5 (48 mg/kg). One foetus had hydrocephalus and two foetuses had palatoschisis (one of which was a runt). These findings may be test substance related. The presence of one runt in groups 2, 3 and 4, respectively, was considered to be incidental.

Besides the mentioned foetus with hydrocephalus in group 5, one foetus in group 4 with hydrocephalus internus was found during visceral investigations by Wilson Technique. This single finding was considered to be incidental because of isolated appearance of foetuses with hydrocephalus internus in the historical background data of this rat strain employed. The skeletal investigations of foetuses for anomalies resulted in similar findings of nonspecific conventional variations in all groups. The comparison of the stage of skeletal development yields an increased incidence of incompletely or still absent ossifications of phalangeal nuclei and calcanea in the foetuses of group 4 (24 mg/kg) and group 5 (48 mg/kg). This result corresponded to the reduced body weights of foetuses.

In conclusion, mean body weight loss was seen in treated dams at the beginning of the treatment period (days 6-8) from 24 mg/kg bw/day. A dose related and biologically significant body weight gain reduction of -18% versus control was observed at the 12 mg/kg bw/day dose level.Mean bodyweight gain recovered thereafter. Mean food consumption was reduced in the 24 and 48 mg/kg bw/day from days 6 -11 and days 11- 16. Embryo/foetal toxicity was evident at 24 and 48 mg/kg from the following observations: decreased total number of live foetuses per dam, increased numbers of early and late resorptions, decreased foetal bodyweight and incomplete ossification of phalangeal nuclei and the absence of ossification in calcanea. At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which one was a runt. A single incidence of hydrocephalus internus was also apparent at the 24 mg/kg bw/day dose level. Given the probably treatment-related incidence in the 48 mg/kg bw/day, relationship to treatment is possible. The maternal NOAEL in this study was 6 mg/kg based on bodyweight gain reduction during early treatment at 12 mg/kg. The developmental NOAEL was 12 mg/kg based on reduced foetal bodyweight, increased post implantation loss and increased incidence foetal malformations at 24 and 48 mg/kg.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Dec 1990 to 04 Mar 1991; amendment of the original report issued in 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

1981

Deviations:

yes

Remarks:

Analysis of concentration, homogeneity and stability indicated a wide range of values

Qualifier:

according to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Version / remarks:

1982

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 14 to 18 weeks
- Weight at study initiation: 2.9 to 4.3 kg
- Housing: The female rabbits were housed individually in metal cages with perforated bottoms (dimensions: 540 mm x 580 mm x 400 mm). The cages were suspended above excrement removal trays which were changed three times weekly.
- Diet: ad libitum
- Water: Tap water was available ad libitum from plastic water bottles attached to each cage
- Acclimation period: At least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 20 Dec 1990 to 04 Mar 1991

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: The test substance was weekly prepared as a suspension in the vehicle. Separate preparations were made for each dose level.

VEHICLE
- Carboxylrnethylcellulose
- Amount of vehicle: 1%

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Separate samples from top, middle, and bottom of the test substance formulations prepared for groups 2 to 4 during the first week of treatment were taken together with a reference sample of the control item and analysed for determination of homogeneity. In addition, samples were taken from the test substance formulations prepared for groups 2 to 4 during the last week of treatment together with a reference sample of the control item and analysed for determination of concentration.

Details on mating procedure:

- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: Sperm in vaginal smear referred to as day 0 of pregnancy
- Females that successfully completed coitus received an intravenous injection of luteinising hormone to ensure ovulation.

Duration of treatment / exposure:

13 days: GD 6 to 18

Frequency of treatment:

Once daily

Duration of test:

28 days

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Intermediate dose

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

18

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on the results of a dose range-finding study.

Maternal examinations:

CLINICAL OBSERVATIONS:
- Time schedule: All animals were examined at least once daily for signs of ill health or overt signs of toxicity and each finding was recorded.

BODY WEIGHT:
- Time schedule for examinations: The body weight of each inseminated female rabbit was recorded and evaluated for days 0, 6, 9, 12, 15, 19, 24, and 28 post-coitum.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption: The food consumption of each inseminated female rabbit was recorded for days 0 to 2, 2 to 4, 4 to 6, 6 to 9, 9 to 12, 12 to 15, 15 to 17, 17 to 19, 19 to 21, 21 to 24, 24 to 26, and 26 to 28 post-coitum.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes

POST-MORTEM EXAMINATIONS:
- Sacrifice: On day 28 post-coitum, the surviving female rabbits were sacrificed by intravenous injection of Eutha 778, dissected, and examined macroscopically for pathological changes. Any abnormalities were recorded. Animals found dead and females showing signs of abortion, were also necropsied and examined for pathological changes.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea
- Number of implantations
- Number of early resorptions
- Number of late resorptions

Fetal examinations:

- External foetal abnormalities
- Individual foetal weight
- Foetal sex
- Skeletal or visceral foetal abnormalities
- The heads were removed from approximately half of the foetuses, fixed in Bouin's fluid, and examined for visceral abnormalities. In the remaining foetuses, a gross examination of the brain for visceral defects was performed by a mid-coronal cut.

Statistics:

For body weight, body weight change, and food consumption, the Levene's test for homogeneity of variance was performed followed by one-way Analysis of Variance. In case of significant results for the ANOVA (p < 0.01 or p < 0.05), the Dunnett's test for multiple group comparisons (p < 0.05) was performed. For litter weights, the Analysis of Variance was performed with one factor TREATMENT followed by the Student-Newman-Keuls test for multiple group comparisons. For number of corpora lutea, number of implantations, preimplantation loss, number of live foetuses, total number of intra- uterine deaths, post implantation loss, foetal weight (overall, males, and females), and mean proportion of male foetuses, the Analysis of Variance was performed with one factor TREATMENT - based on taking the ranks of the variables - and followed by the Student-Newman-Keuls test for multiple group comparisons. For the total number of litters with malformed foetuses the Wilcoxon Rank-Sum test was used to compare each treated group against the control. The statistical evaluation was performed with the standard software package SAS (Statistical Analysis System) release 6.03 excluding the analysis for body weight, body weight change, and food consumption which was performed with the statistical package of the on-line data collection system TERASYS.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Low water consumption was observed in animals of all groups but at increased incidences in group 4 (50 mg/kg). As six of the nine animals affected in this dose group showed this type of finding on one or more of the first days of the treatment period, this finding is considered attributable to treatment. Two animals each in groups 2 (2 mg/kg) and 4 (50 mg/kg) aborted and were therefore killed on days 27, 20, 19, and 19 post-coitum, respectively. One animal did not show any relevant clinical signs prior to abortion. At necropsy, autolytic foetuses and placental as well as foetal remnants were found in this animal. In another animal, low water consumption, few faeces, and diarrhoea were observed on some of the days prior to abortion, but necropsy did not reveal any relevant findings. Another animal had previously shown low water consumption but did not show any relevant clinical findings on the days prior to abortion and no remarkable observations were recorded at necropsy. In another animal, no relevant clinical signs or necropsy findings were detected. As the incidences of abortions did not show any dosage-relationship and as similar incidences have earlier been observed in this strain of rabbits, the abortions in this study cannot be related to treatment and are therefore considered incidental. Three females of group 2 (2 mg/kg) and one female each of groups 3 (10 mg/kg) and 4 (50 mg/kg) showed 100 per cent intra-uterine deaths at necropsy. These findings cannot be related to treatment as they do not show any dosage-relationship and as similar numbers are known from control groups of other studies. Further relevant clinical observations were not made in the dose groups or the control group of this study.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One animal each in groups 3 (10 mg/kg) and 4 (50 mg/kg) were found dead on days 14 or 22 post-coitum. In these animals, clinical signs as few faeces or low water consumption were detected on the day of their death or some single days before. The numerous necropsy findings detected mainly or exclusively in the thoracic organs of these animals did not show any treatment-relationship and therefore these mortalities are considered incidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the dose groups and the control group, inter-group and intra group differences of group mean body weight change were observed. In group 4 (50 mg/kg), a clear group mean body weight loss was observed from day 6 to 9 post-coitum. The difference to the control group where a clear increase of the group mean body weight was observed was statistically significant. This finding is considered to be treatment related. From day 19 to 24 post-coitum, this difference to the control group was partly compensated. In group 3 (10 mg/kg), group mean body weight change was lower than in the control group throughout the dosage period, especially from days 6 to 9 post-coitum. This finding was not statistically significant. In group 2 (2 mg/kg), group mean body weight change was similar to the control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

In group 4 (50 mg/kg), mean daily food consumption was markedly and statistically significantly reduced from day 6 to 9 post-coitum when compared with the control group. Accordingly, mean daily food consumption from day 6 to 19 post-coitum was reduced in this dose group, too. This finding is considered to be treatment related. From day 21 to 28 post-coitum, mean daily food consumption was slightly higher than in the control group but food consumption was never fully compensated. In the other dose groups, mean daily food consumption also showed differences to the control group but these differences were only small and did not show any treatment relationship. These differences are therefore considered incidental.

Food efficiency:

no effects observed

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

effects observed, treatment-related

Description (incidence and severity):

Low water consumption was observed in animals of all groups but at increased incidences in group 4 (50 mg/kg). As six of the nine animals affected in this dose group showed this type of finding on one or more of the first days of the treatment period, this finding is considered attributable to treatment.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Necropsy on day 28 post-coitum revealed findings in various organs of animals from all groups but all these findings were irrelevant and did not indicate any effect of treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Number of abortions:

effects observed, non-treatment-related

Pre- and post-implantation loss:

effects observed, non-treatment-related

Description (incidence and severity):

Preimplantation loss was highest in group 3 (10 mg/kg) and lowest in group 2 (2 mg/kg). But as the results of these two dose groups as well as those from the high dose group and the control group are within the range of historical control data, the results obtained are considered incidental.
Post implantation loss, if calculated from animals with live foetuses only, was similar and low in all groups. If animals with total intra-uterine deaths are included into calculation (three animals in group 2 and one each in groups 3 and 4), post implantation loss is highest in group 2 (2 mg/kg), accordingly. As the observed results are within the range of historical control data and as a dosage-relationship could not be detected, these findings cannot be related to treatment and are therefore considered incidental.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Changes in number of pregnant:

no effects observed

Other effects:

effects observed, treatment-related

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

not examined

External malformations:

effects observed, treatment-related

Description (incidence and severity):

Malformations in the tail seen in the high dose group

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

In the control group, three foetuses from three litters showed external or skeletal malformations as kinked tail, fused ribs, or malpositioned digits. In the low dose group (2 mg/kg), one foetus was viscerally malformed showing dysplasia of the retina. In the mid dose group (10 mg/kg), six foetuses (one of them dead) from three litters, showed external/visceral and skeletal malformations. Some of these malformations concerned ribs and vertebrae, the position of hindlimbs, the tail and the abdominal wall. The outcome of this group was dominated. Four of the six affected foetuses were from this single litter (positions 1-4 in right horn of uterus). One of these four was found dead at necropsy and two further had extremely low body weights (21.5 and 22.8 g compared to the group mean of 40.8 g). These three foetuses were found with omphalocele, umbilical hernia and arthrogryposis. The remaining three affected foetuses of this group were found with skeletal malformations only.
In the high dose group (50 mg/kg), 15 foetuses from seven litters were malformed. These malformations were found in sternebrae and ribs, vertebral column, hindlimbs.
Due to the types and incidences of malformations, and when compared with the concurrent and historical control data, a treatment-related effect is concluded for the high dose group. A significant incidence of malrotated hindlimbs was recorded in the high dose animals. There was one incidence of this malformation in the 10 mg/kg bw/day group also. This malformation was not seen in the historical control data presented and association with treatment cannot be excluded at present.

Visceral malformations:

effects observed, treatment-related

Description (incidence and severity):

Malformations in the kidney seen in the high dose group.

Key result

Dose descriptor:

NOAEL

Remarks:

(=NOEL)

Effect level:

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

Based on:

test mat.

Sex:

not specified

Basis for effect level:

skeletal malformations

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

external: tail

skeletal: rib

skeletal: vertebra

skeletal: hindlimb

visceral/soft tissue: urinary

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

developmental effects in the absence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Table 1. Group mean maternal body weight change

	Group mean maternal body weight change (gms)
	0 mg/kg	2 mg/kg	10 mg/kg	50 mg/kg
	N=16	N=13	N=15	N=13
Days 0-6	356.95±112.63	291.84±139.79	318±105	278.5±117
Days 6-9	70.98±54	71±65.35	55±91	-36.94**±118
Days 9-12	102.46±44.7	58.26±107.76	69.4±70	121±80
Days 12-15	71.51±58.64	80.41±167	83±54 (14)	47±82
Days 15-19	104.96±81.99	71.72±93	64±94 (14)	91±110
Days 19-24	96.18±79.75	112.5±128 (12)	135±62 (14)	148±60) (10)
Days 24-28	96.86±90	101±88.6 (11)	87±70 (14)	47±156 (10)
Days 0-28	899.89±247.22	790±323 (11)	833±235.45 (14)	765±187 (10)
Days 6-19	349.90±103.33	281±194 (13)	293.5±148 (14)	222±176 (13)

*p<0.05;** p<0.01

Table 2. Data on reproduction (including animals with live foetusus)

Parameter		0 mg/kg	2 mg/kg	10 mg/kg	50 mg/kg
Number of mated females		18	18	18	18
Number of non-pregnant females		2	2	2	4
Number of pregnant females which were found dead				1	1
Number of females which aborted (therefore killed)			2		2
Number of females with 100% intra-uterine deaths			3	1	1
Number of females with live foetuses at necropsy		16	11	14	10
(=numbers used for calculations)
Number of corpora lutea:	per group	201	139	160	106
	mean per dam	12.6	12.6	11.4	10.6
Number of implantations:	per group	120	100	87	67
	mean per dam	7.5	9.1	6.2	6.7
Preimplantation loss:	per group	81	39	73	39
	mean per dam	5.1	3.5	5.2	3.9
Live foetuses:                     per group		110	90	75	60
mean per dam		6.9	8.2	5.4	6.0
% of implantations		91.3	87.9	87.5	91.3
% males		58.6	52.6	53.3	53.6
Dead foetuses		1	0	2	1
Early resorptions:                 per group		7	8	9	6
mean per dam		0.4	0.7	0.6	0.6
Late resorptions:                  per group		2	2	1	0
mean per dam		0.1	0.2	0.1	0.0
Total pos-timplantation loss: per group		10	10	12	7
mean per dam		0.6	0.9	0.9	0.7
% of implantations		8.7	12.1	12.5	8.7
Mean weight of live foetuses:		38.5	37.4	40.8	38.1

Table 3. Data on reproduction (foetal abnormalities)

Parameter	0 mg/kg	2 mg/kg	10 mg/kg	50 mg/kg
Number of foetuses examined externally (no. of dead foetuses) Number of foetuses examined viscerally Number of foetuses examined skeletally	110 (1)   110 110	90   90 90	75 (2)   73 73	60 (1)   59 59
Malformations
No. of foetuses withexternal/visceral malformations(no. litters affected)	2 (2)	1 (1)	2 (1)	7 (5)
No. of foetuses withskeletal malformations (no. litters affected)	1 (1)	0 (0)	3 (3)	13 (7)
Totalno. of foetuses withmalformations -(no. litters affected)	3 (3)	1 (1)	5 (3)	15 (7*)
-Average % malformed foetuses	2.7	1.1	6.1	25
-% of litters affected	18.8	9.1	21.4	70
Variations
No. of foetuses with external/visceral variations (no. litters affected)	3 (2)	3 (1)	3 (2)	2 (2)
No. of foetuses with skeletal variations (no. litters affected)	110 (16)	88 (11)	72 (14)	58 (10)

Conclusions:

The NOAEL for maternal toxicity is considered to be 10 mg/kg bw/day and for developmental effects is 2 mg/kg bw/day based on the presence of skeletal malformations in fetuses at the mid and high dose level.

Executive summary:

In a developmental toxicity study in accordance with OECD TG 414 and GLP conditions, groups of 18 inseminated female New Zealand White rabbits received the test substance by oral gavage at dosages of 2, 10, or 50 mg/kg/day for thirteen consecutive days from day 6 to 18 post-coitum. A further group of 18 inseminated female rabbits of the same strain which received the vehicle (1% carboxymethylcellulose) over the same period served as the control group. The animals were sacrificed on day 28 post-coitum.
Results showed, at 50 mg/kg/day, treatment-related maternal body weight gains and food consumption decreased throughout the study, especially days 6 through 9, and low water consumption was observed with increased incidence. Incidental occurrences included one death, two abortions, and random differences in pre- and post-implantation losses. The mean number of foetuses per dam, foetal sex distribution, and mean foetal weight did not indicate any treatment-related effects. No treatment-related mortalities were observed. Necropsy of dams was unremarkable. At the same dose level,hese findings were accompanied by increased total external/visceral or skeletal malformations in litters which were statistically significant when compared with the concurrent control group. Fifteen foetuses in seven litters were considered malformed. The incidence and type of skeletal and external/visceral malformations were increased at this dose level
At 10 mg/kg/day, maternal toxicity was observed as decreases in body weight gains throughout the dosage period, especially during the first three days of dosing. Food consumption was also lower. These parameters were not statistically significant and could not be attributed to treatment with certainty. One death, but no abortions or meaningful differences in pre- and post-implantation losses of foetuses, was among the incidental occurrences reported. Necropsy of dams was unremarkable. The incidence of external/visceral or skeletal malformations observed were not statistically significantly increased over the concurrent control values. An incidence of malrotated hiindlimps was recorded that was not seen in historical control data, and as such, association with treatment could not be excluded. No other developmental effects were detected.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

2 mg/kg bw/day

Study duration:

subchronic

Species:

rabbit

Quality of whole database:

A guideline study (comparable to OECD 414) performed in compliance with GLP

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 test in rabbits

In a developmental toxicity study in accordance with OECD TG 414 and GLP conditions (Muller 1991), groups of 18 inseminated female New Zealand White rabbits received the test substance by oral gavage at dosages of 2, 10, or 50 mg/kg/day for 13 consecutive days from day 6 to 18 post-coitum. A further group of 18 inseminated female rabbits of the same strain which received the vehicle (1 % carboxymethylcellulose) over the same period served as the control group. The animals were sacrificed on day 28 post-coitum.

Results showed, at 50 mg/kg bw/day, treatment-related maternal body weight gains and food consumption decreased throughout the study, especially days 6 through 9, and low water consumption was observed with increased incidence. Incidental occurrences included one death, two abortions, and random differences in pre- and post-implantation losses. The mean number of foetuses per dam, foetal sex distribution, and mean foetal weight did not indicate any treatment-related effects. No treatment-related mortalities were observed. Necropsy of dams was unremarkable. These findings were accompanied by increased total external/visceral or skeletal malformations in litters which were statistically significant when compared with the concurrent control group. Fifteen foetuses in seven litters were considered malformed. The incidence of litters and/or foetuses with malformations observed in this study was within the range of historical control data. The incidence and type of skeletal and external/visceral malformations were increased at 50 mg/kg bw/day; however, the types of malformations were similar to those observed in the historical controls.

At 10 mg/kg bw/day, maternal toxicity was observed as decreases in body weight gains throughout the dosage period, especially during the first three days of dosing. Food consumption was also lower. These parameters were not statistically significant and could not be attributed to treatment with certainty. One death, but no abortions or meaningful differences in pre- and post-implantation losses of foetuses, was among the incidental occurrences reported. Necropsy of dams was unremarkable. The incidence of external/visceral or skeletal malformations was not statistically significantly increased over the concurrent control values, but as the malformation was not seen in historical control data, association with treatment could not be excluded. No developmental toxicity was detected.

At 2 mg/kg bw/day, no maternal toxicity (although two incidental abortions were reported) and no developmental toxicity were observed.

In conclusion, administration of the test substance to pregnant rabbits at the dose level of 50 mg/kg bw/day resulted in body weight loss and reduced food consumption early during the treatment period. Reproduction parameters and mean foetal body weights were not affected at any dose level. An increased incidence of foetal malformations was obtained at the highest dose of 50 mg/kg bw/day. In addition, the increased incidence of skeletal malformations at 10 mg/kg bw/day may be treatment-related. The occurrence of a single incidence of malrotated hind limbs was noted at 10 mg/kg bw/day. The NOAEL for maternal toxicity is considered to be 10 mg/kg bw/day and for developmental effects is therefore, 2 mg/kg bw/day.

 

Another study (Becker 1986), complying to GLP and OECD TG 414, investigated the effect of the test substance when administered by gavage in Chinchilla rabbits. This study showed a treatment-related decrease in maternal weight gain in the high dose (50 mg/kg bw/day), decrease in food consumption and a significantly increased post implantation loss. At the middle dose (10 mg/kg bw/day) an significant implantation loss was recorded. Also in this study, the maternal NOAEL was considered to be 10 mg/kg bw/day and the developmental NOAEL was considered to be 2 mg/kg bw/day.

 

Developmental toxicity test in rats

A developmental toxicity study in rats was performed in accordance with OECD TG 414 and GLP principles (Becker 1985), to determine the potential of the test substance to induce structural and/or other anomalies in the foetus which may arise from the exposure of the mother during pregnancy. The test substance was administered orally by intubation once daily to mated female Wistar rats (outbred, SPF-quality) from day 6 through day 15 post coitum. Animals were treated at dose levels of 0, 6, 12, 24 and 48 mg/kg bw/day by oral gavage at a constant dosing volume of 10 mL/kg bw. The vehicle that was used in this study was CMC. Each group consisted of 25 mated female rats. On day 21 post coitum, all females were sacrificed and the foetuses removed by caesarean section. The investigations of females/dams and foetuses were performed in accordance with international recommendations. All parameters recorded were evaluated and reported.

Results showed that no death occurred and no substance related signs or symptoms were observed. At necropsy, no test substance related findings were evident in any female of any group. The oral administration of the test substance caused loss of body weight in the dams of group 5 (48 mg/kg bw/day), from the first to second and from the second to third day of treatment. Thereafter the body weight gain was similar to that of the vehicle control group. The reason that the body weights remained below that of the vehicle control group was as noted as in group 4 (24 mg/kg bw/day), primarily the consequence of the lower number of foetuses per dam. A dose related and biologically significant body weight gain reduction of -18 % versus control was observed at the 12 mg/kg bw/day dose level. The mean food consumption of groups 4 and 5 was significantly reduced (dose-related) during the treatment period in comparison to that of the vehicle control group. No significant or test substance related differences in body weight gain and food consumption were noted between the vehicle control group and group 2 (6 mg/kg bw/day) or 3 (12 mg/kg bw/day). The evaluations of the reproduction data resulted in dose related significantly increased post implantation losses of 22.2 % in group 4 (24 mg/kg bw/day) and 34.2 % in group 5 (48 mg/kg bw/day). In the vehicle control group and in groups 2 (6 mg/kg bw/day) and 3 (12 mg/kg bw/day) losses of 5.0 %, 4.2 % and 2.9 % were noted, respectively. In the foetuses, slight but significantly reduced mean body weights of 8.3 % were noted in groups 4 and 5 when compared to that of the vehicle control group. During the external investigations of foetuses, three foetuses of different litters/dams with anomalies were found in group 5 (48 mg/kg bw/day). One foetus had hydrocephalus and two foetuses had palatoschisis (one of which was a runt). These findings may be test substance related. The presence of one runt in groups 2, 3 and 4, respectively, was considered to be incidental.

Besides the mentioned foetus with hydrocephalus in group 5, one foetus in group 4 with hydrocephalus internus was found during visceral investigations by Wilson Technique. This single finding was considered to be incidental because of isolated appearance of foetuses with hydrocephalus internus in the historical background data of this rat strain employed. The skeletal investigations of foetuses for anomalies resulted in similar findings of nonspecific conventional variations in all groups. The comparison of the stage of skeletal development yields an increased incidence of incompletely or still absent ossifications of phalangeal nuclei and calcanea in the foetuses of group 4 (24 mg/kg bw/day) and group 5 (48 mg/kg bw/day). This result corresponded to the reduced body weights of foetuses.

In conclusion, mean body weight loss was seen in treated dams at the beginning of the treatment period (days 6-8) from 24 mg/kg bw/day. A dose related and biologically significant body weight gain reduction of -18 % versus control was observed at the 12 mg/kg bw/day dose level. Mean bodyweight gain recovered thereafter. Mean food consumption was reduced in the 24 and 48 mg/kg bw/day from days 6 -11 and days 11- 16. Embryo/foetal toxicity was evident at 24 and 48 mg/kg bw/day from the following observations: decreased total number of live foetuses per dam, increased numbers of early and late resorptions, decreased foetal bodyweight and incomplete ossification of phalangeal nuclei and the absence of ossification in calcanea. At the highest dose one foetus was noted with a hydrocephalus and two foetuses had a palatoschisis (also reported as cleft palate), of which one was a runt. A single incidence of hydrocephalus internus was also apparent at the 24 mg/kg bw/day dose level. Given the probably treatment-related incidence in the 48 mg/kg bw/day, relationship to treatment is possible. The maternal NOAEL in this study was 6 mg/kg bw/day based on bodyweight gain reduction during early treatment at 12 mg/kg bw/day. The developmental NOAEL was 12 mg/kg bw/day based on reduced foetal body weight, increased post implantation loss and increased incidence foetal malformations at 24 and 48 mg/kg bw/day.

 

Another developmental toxicity study (Machera 1994), performed in rats, was available from published data. The effects were apparent at all doses and were considered treatment related. Again here the test material induced malformations to the growing fetuses. No NOAEL could be established in this study, neither for maternal nor for embryo-/foetal toxicity.

Justification for classification or non-classification

Based on the available information the substance is classified as Repr 1B for reproductive toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.