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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 1990 to November 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with no major deficiencies.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
May to June 1990
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, meets generally accepted scientific method and is described in sufficient detail. Test substance analytical monitoring was performed. No statistical analysis of the results, only external observation performed.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
no guideline required
Principles of method if other than guideline:
No method specified, this study was a range-finding study (OECD 414)
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Crl:CD(SD)BR
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK).
- Age at study initiation: 10 to 15 weeks
- Weight at study initiation: 185-245 g at the start of the study.
- Fasting period before study:
- Housing: females mated were individually caged
- Diet and water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): solutions of the test article in distilled water were prepared daily. Nitrogen gas was bubbled through all formulations to reduce the dissolved oxygen content of the water.

- Storage temperature of food: formulations were stored at room temperature in containers purged with nitrogen.

Analytical verification of doses or concentrations:
yes
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: no data
- Length of cohabitation: overnight
- 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 were delivered to the study laboratory on day 2 of gestation
Duration of treatment / exposure:
From day 6 to 15 of gestation, inclusive.
Frequency of treatment:
Daily.
Duration of test:
untill day 20 of gestation
No. of animals per sex per dose:
seven females per dose
Control animals:
yes, concurrent vehicle
Maternal examinations:
CAGE SIDE OBSERVATIONS:
All females were examined twice daily to detect any which were dead or moribund.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: all females were examined daily during gestation. Any abnormalities of appearance or behaviour or other signs of reaction to treatment or ill health were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: on days 3, 6, 7, 8, 9, 12, 15, 18 and 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
Pregnancy status, number of corpora lutea, number and intrauterine position of implantations subdivided into: live foetuses, early intrauterine deaths, late intrauterine deaths and dead foetuses.
Fetal examinations:
- External examinations: Yes
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:

- Morbidity:
On day 19 of gestation, one female in the high dose group died and one was killed in extremis, due to severe bodyweight loss and reduced food intake. At necropsy one female showed a yellow fluid in the abdominal cavity and jejunum, and all implantations were dead, and the second one showed no gross abnormalities and the majority of implantations had died before necropsy.

- Clinical signs:
In addition to the two females killed in extremis, two others in the High dose group were observed to be hyperactive since Day 9 of gestation. Other changes were limited to commonly occurring clinical signs.

- Bodyweight:
Medium dose group and high dose group were characterised by a reduced bodyweight gain in the same order of magnitude (-20%) compared with animals from the control group.

- Food intake: Food consumption was lower in the high dose group (-25%) than in the other groups, control group included. Food intake of the low dose group was only marginally lower than controls.
Dose descriptor:
NOAEL
Effect level:
22.5 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
45 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Remarks:
(highest dose tested)
Effect level:
67.5 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: developmental toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:
no effects

Details on embryotoxic / teratogenic effects:
- Uterine implantation (Table 1): The only non-pregnant animal was in the low dose group. Pre-implantation is expected to occur before the start of dosing (Day 6). Variations in mean preimplantation loss between treatment groups cannot be affected to treatment. Post-implantation loss changes were not related to the test item.

- Foetal data:
Mean number of foetus per females was between 11.7 (low dose group) to 13.6 (high dose group), sex ratio was in the range of the historical data (from 40 to 58% of males). Foetus weight was slightly lower for pups from the two highest groups (10%) but it is not statistically significant (test, not performed by the laboratory, additional analysis). Litter weight was comparable to or higher than controls in the low and high dose groups and marginally reduced in the intermediate dose group.
No malformation was seen by external observation of the foetuses. Variations were limited to subcutaneous haemorrhagic areas or pale colouration. These findings are commonly observed in this strain of rat.
Abnormalities:
not specified
Developmental effects observed:
not specified

No malformations were observed. 

The results allowed the choice of  suitable dose rates for the main study.




Table 1: Implantation data of female rat daily administered with THPS at  3 dose levels (low: 22.5 mg/kg bw, medium: 45 mg/kg bw,  high: 67.5 mg/kg  bw, main ingredient).

 

Control     

Low dose     

Medium dose      

High dose

N females                        

7

7

7

7

N pregnant                      

7

6

7

7

Female with pre-implantation loss

1

3

4

4

Mean pre-implantation    loss (%)

1.0

8.9*

4.0

4.2

Female with post-implantation loss

2

0

2

1**

Mean post-implantation  loss (%)

2.2

0

2.2

1.4

* one female lose 33.3% of implantation
** Two females died or were killed on Day 19 and were discounted.


Conclusions:
Under the test conditions of this study, treatment with main ingredient at 67.5 mg/kg bw/d (as main ingredient) elicited severe maternal toxicity. Maternal toxicity was also observed at the medium dose (45 mg/kg bw/d) but in a lesser extent. No effects to foetuses could be related to treatment. The low dose level was recommended to be chosen below 30 mg/kg/day (as main ingredient) as this was not a "no-effect level".
Executive summary:

Female rats, Crl:CD(SD)BR strain, of 10 to 15 weeks old (185 - 235 g) were mated with untreated male rats of the same strain (range finding study equivalent to OECD 414).

Seven females per dose were orally (gavage) administered during day 6 to 15 of gestation, inclusive, at 30, 60 or 90 mg/kg bw (active substance), or 22.5, 45 and 64.8 mg/kg bw (main ingredient, THPS 75%), and one control group. All animals were maintained until day 20 of gestation, when they were killed and their uterine contents examined. Mated females were caged individually and had free access to a pelleted diet and filtered mains water.

 

Animals were dosed daily with freshly prepared test solution, by dilution of the test item with distilled water.

 

Viability, mortality and clinical signs were done every day. Bodyweight and food intake were monitored 9 times from days 3 to 20. At the end of the study, females were killed and a necropsy was performed with macroscopic observation. Moreover, pregnancy status, uterine content and foetus aspects were examined. The following data were recorded: number of corpora lutea, number and intrauterine position of implantations, subdivided into live foetuses, early and late intrauterine deaths, dead foetuses. Live foetus were killed, weighted, sexed and examined externally.

After external examined, the foetuses were discarded.

 

Treatment with main ingredient at 67.5 mg/kg bw/d elicited severe maternal toxicity. Maternal toxicity was also observed at the medium dose (45 mg/kg bw/d main ingredient) but in a lesser extent. No effects to foetuses could be related to treatment. The low dose level was recommended to be chosen below 30 mg/kg/day (22.5 mg/kg bw/d, as active ingredient) as this was not a "no-effect level".

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
No guideline available at the time of the study, but complies with the requirement of the OECD 414 Guideline (22/01/2001). The treatment period was only from day 6 to day 15 of gestation.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
EC Number:
259-709-0
EC Name:
Tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Cas Number:
55566-30-8
Molecular formula:
C4H12O4P.1/2O4S
IUPAC Name:
tetrakis(hydroxymethyl)phosphonium sulphate(2:1)
Test material form:
other: liquid stored at rrom temperature
Details on test material:
- Name of test material (as cited in study report): THPS-75
- Substance type: colourless liquid
- Storage condition of test material: stored at ambient temperature in the dark.

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR, females
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK).
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation: 217-267 g at mating
- Fasting period before study:
- Housing: before mating, five by sex in a cage, for mating, one male with one or two females and females mated were individually caged
- Diet and water ad libitum
- Acclimation period: at least 12 days before mating

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C
- Humidity: 40-70%
- Air changes: 15 per hour
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From 31 May to 9 July 1990

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
solutions were prepared daily, and stored in containers purged with nitrogen before use, to remove any dissolved oxygen

VEHICLE
- Concentration in vehicle:
Expressed as active substance: 3, 6 and 12 mg/ml,
Expressed as main ingredient: 2.3, 4.5 and 9.1 mg/ml.
- Amount of vehicle: 5 ml/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples from each dose group were analysed by iodine titration on the first and last day of dosing.
Measured concentrations were 95 to 100 % of the nominal concentrations.
Details on mating procedure:
- Impregnation procedure: cohoused
- M/F ratio per cage: one male for one or two females
- Length of cohabitation: mating period: 6 days over a period of 8 days.
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From day 6 to day 15 of gestation.
Frequency of treatment:
Daily.
Duration of test:
Animals maintained until day 20 of gestation at which time they were killed.
No. of animals per sex per dose:
24 females per group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Based on the results of the range finding study (development tox. V2 1991b Bark_rat_RF)

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Daily.

DETAILED CLINICAL OBSERVATIONS: Daily.

BODY WEIGHT: Recorded on days 0, 6, 7, 8, 9, 12, 15 and 20 of gestation

FOOD CONSUMPTION: Recorded individually on days 6, 7, 8, 9, 12, 15 and 20 of gestation

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 20
- Organs examined: ovaries and uteri
Ovaries and uterine content:
Examinations included:
Number of corpora lutea
Number of gravid females and uterine weight. The pregnancy status was assessed by ammonium sulphide staining.
Number, position (uterine horn side and rank from 1 to 10) and living status (live/death, early/late death) of implantations.

Fetal examinations:
Litter size, number of dead foetuses, individual foetal weight, sex ratio.
Skeleton: Observations were performed approximately on one half of the foetuses in each litter. After visceral examination and removal, the carcass processed to stain the ossified skeleton. Observations were performed using low power binocular magnification.
Soft tissue: The remaining foetuses were partially decalcified and stained. The head was removed and sections
were examined, and the remaining body was examined by dissection using low power binocular magnification.

Foetal abnormalities were recorded as malformation (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities).
Statistics:
Analysis of Variance was performed (Levene’s test) for equality of variances between groups. Comparisons between each treated group and control was made using Dunnett’s test. If the data were not suitable for this procedure, non-parametric methods (Kurskal-Wallis’,
Wilcoxon’s Rank-Sum and Terpstra-Jonkcheere’s tests) were performed to compare variance, mean and dose-response.
Historical control data:
Historical control data were issued from the 6 rat teratology studies immediately preceding the current study.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes. Remark: (See Table 1)

Details on maternal toxic effects:
All females were alive at the end of the study. Recurrent clinical effects observed in each group, control group included, were hair loss, fur staining, sores/lesions (head, neck, ears). In addition, animals from the medium (22.3 mg/kg bw) and high (45.3 mg/kg bw) dose groups showed excessive salivation, respectively 5 and 17 animals, and three animals dosed at 45.3 mg/kg bw were thin and showed piloerection and hunched posture from day 15 of gestation.
Necropsy revealed gastro intestinal lesions in one female from the control group and in four in both the medium dose group and the high dose group. Lesions were considered as minor (thickening of the mucosa surface of the stomach or duodenum), except for two out of the three animals affected by severe clinical signs from the high dose group, where gastro intestinal lesions were more severe (duodenal ulcer/abnormal ingesta).
Bodyweight gain and food consumption were reduced by 4 to 16%, compared with control animals, or 3 to 6% if data from the three severely affected females were discounted.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEL
Effect level:
45.3 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
22.7 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects. Remark: (See Table 2&3)

Details on embryotoxic / teratogenic effects:
All the parameters observed in the treated groups (pregnancy rate, implantation loss, number of living foetus, sex ratio…) were in the same order than those recorded in the control group, and were within the range of the historical findings. However, in the high dose group (45.3 mg/kg bw) mean foetal weight was lower than control; this is entirely attributable to the three females which showed severe clinical signs and weight loss from day 15 to 20 of gestation. If data from these females are excluded, the high dose mean foetal weight is similar to that recorded for control foetuses.
Foetuses with malformations were observed in the low dose group (11.3 mg/kg bw) and in the high dose group. In the low dose group, two foetuses from two different dams, exhibited hydrocephaly or hydronephrosis. For the high dose group, one foetus showed a haemorrhagic eye and the second anophtalmia, eye socket reduced in size, cervical vertebral arches fused. This latest foetus came from one of the three females severely affected. These malformations have been observed in historical control litters, and part of them is known to occur spontaneously in this strain of rat (hydronephrosis).
There was a statistical significant increase in the number of foetuses showing extra thoraco-lumbar ribs in the high dose group. The number of affected litters was also higher in this dose group than in the control group, respectively 14 and 6 litters. Supernumerary ribs occur spontaneously in most strains of rats and their frequency can be increased by many extrinsic agents.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
45.3 mg/kg bw/day
Based on:
act. ingr.
Remarks:
THPS
Basis for effect level:
other: developmenbtal toxicity, increase in supernumerary rib and eye defects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1:Maternal effects

Parameter

control data

low dose

medium dose

high dose

dose-response
+ / -

Historical1

study

Number of dams examined

153

24

24

24

24

-

Clinical findings during application of test substance

ND

· Hair loss, fur staining, sore/lesions (head, neck, ears).

· Hair loss, fur staining, sore/lesions (head, neck, ears).

· Hair loss, fur staining, sore/lesions (head, neck, ears),

· Salivation.

· Hair loss, fur staining, sore/lesions (head, neck, ears)

· Salivation,

· Thin, piloerection, hunched posture

+

Mortality of dams (%)

ND

0

0

0

0

 

Abortions

ND

0

0

0

0

 

Body weight gain (%)

day 0-20 (end of test)

day 6-15 (dosing period)

ND

 

61.3

20.8

 

60.9

19.9

 

61.9

21.1

 

48.5**/55.5a*

14.2**/16.4a**

+

Food consumption (d/rat/day)

day 0-20 (end of test)

day 6-15 (dosing period)

day 15-20 (post dosing)

ND

 

25

24

31

 

26

24

31

 

26

25

31

 

24/25a

22***/23a*

26/30a

+

Pregnancies (%)

95 (83.3-100)

96

92

92

100

 

Necropsy findings in dams dead before end of test

ND

-

-

-

-

 

1Historical control data were issued from the 6 rat teratology studies immediately preceding the current study.

ND: No Data

aExcluding three females showing severe clinical signs

Statistical analysis: *: 0.05>p>0.01; **: 0.01>p>0.001; ***: 0.0001>p

Table 2: Litter response (Caesarean section data)

Parameter

controldata

low dose

medium dose

high dose

dose-response
+ / -

Historical1

study

Corpora lutea (per dam)

15.4 (14.4-16.5)

16.2

15.3

15.4

14.9/15.0a

-

Implantations (per dam)

14 (13.3-15.3)

14.5

14.6

14.0

13.7/13.9a

-

Resorptionsb(per dam)

Early resorption

Late resorption

0.67 (0.4-1.0)

0.62 (0.40-0.80)

0.05(0-0.20)

0.26

0.22

0.04

0.50

0.50

0

0.55

0.55

0

0.29/0.29a

0.29/0.29a

0/0a

-

Total number of foetuses

1937

327

311

296

321/286a

-

Pre-implantation loss (%)

9.38(3.9-19.5

10.7

4.2

8.9

8.4/7.3a

-

Post-implantation loss (%)

4.9 (3.2-7.2)

0

0

0

0/0a

-

Total number of litters

146

23

22

22

24/21a

-

Foetuses / litter

13.3 (12.4-14.5)

14.2

14.1

13.5

13.4/13.6a

-

Live foetuses / litter

13.3 (12.4-14.5)

14.2

14.1

13.5

13.4/13.6a

-

Dead foetuses / litter

0

0

0

0

0

-

Foetus weight (mean, g)

3.44(3.2-3.7)

3.61

3.62

3.64

3.46*/3.61a

-

Foetal male ratio (%)

50.4 (46.6-55.3)

55.4

51.4

51.45

52.0/51.7a

-

1Historical control data were issued from the 6 rat teratology studies immediately preceding the current study.

aExcluding three females showing severe clinical signs,

bResorption = Early resorption (intrauterine death with decidual or placental tissue only) + Late resorption (intrauterine death with embryonic or foetal tissue in addition to placental tissue),

Statistical analysis: *: 0.05>p>0.01

Table 3: Examination of the foetuses

 

Parameter

controldata

low dose

medium dose

high dose

dose-response

+ / -

Historical1

study

 

 

Number of foetus examined for external/visceral abnormalities

1935

327

311

296

321/286a

-

External/Visceral malformations (%)

0.7 (0-3.0)b

Hydrocephaly, anophthalmia, dilatation of aortic arch…

0

0.6b

Hydrocephaly, hydronephrosis

0

0.6b/0.3a

Haemorrhagic eyes, anophthalimia/ Haemorrhagic eyea

-

External/Visceral anomalies (%)

23.4 (8.6-36.7)

26.6

24.4

19.9

24.9/26.2

-

Number of foetus examined for skeletal abnormalities

1281

170

161

153

165/146a

-

Skeletal malformations (%)

0.55(0-1.4)b

Sternebrae fusion, lower jaw bifid, reduced eye socket…

0

0

0

0.6/0a

Reduced eye socket, cervical vertebral arches fused/-a

-

Skeletal anomalies (%)

Extra thoraco-lumbar ribs (%)

92.3 (84-98.2)

ND

91.2

5.9

93.2

6.8

92.8

7.2

92.1/92.5a

20*/20a*

-

+

1Historical control data were issued from the 6 rat teratology studies immediately preceding the current study.

aExcluding three females showing severe clinical signs,

bMalformations were observed on different foetuses from different litters.

Statistical analysis: *: 0.05>p>0.01


NO(A)EL and LO(A)EL for maternal and foetal toxicities are presented below:

 

Maternal toxicity

Foetal  toxicity

NO(A)EL

LO(A)EL

NO(A)EL

Active substance (mg/kg bw)

30

60

60

Main substance (mg/kg bw)

22.7

45.3

45.3

Applicant's summary and conclusion

Conclusions:
Under the test conditions of this study, the LOAEL (maternal toxic effects) was 60 mg/kg bw/ day expressed as active substance or 45.3 mg/kg bw/day expressed as main ingredient, the NOAEL (maternal toxic effects) was 30 mg/kg bw/day expressed as active substance or 22.7 mg/kg bw/day expressed as main ingredient. The NOAEL (embryotoxic/teratogenic effects) was 60 mg/kg bw/day expressed as active substance or 45.3 mg/kg bw/day expressed as main ingredient.
Executive summary:

The teratology study was performed with THPS 75% with female rats according to the OECD Guideline 414. Mated rat females (Crl:CD(SD)BR strain), 11 to 12 weeks old, were orally administered by gavage from days 6 to 15 of gestation, and were observed until day 20 of gestation.Twenty four females were given at one of the three selected dose levels (15, 30, 60 mg/kg bw expressed as active substance, or 11.3, 22.7, 45.3 mg/kg bw expressed as main ingredient) or only with vehicle (distilled water – control group).

Maternal toxicity was assessed by checking mortality, morbidity and clinical signs daily, bodyweight and food consumption at least every two days, and necropsy was performed at Day 20 of gestation. Embryotoxic effect was assessed by examination of uterus content (number of gravid females, number of corpora lutea, implantation status) and observation of foetuses. Foetal abnormalities were recorded as malformation (rare and/or potentially lethal defects) and variations (commonly occurring non-lethal abnormalities) of soft tissue (all foetuses) and skeletal structures (approximately the half of the foetuses).

- Maternal toxicity:

All females were alive at the end of the study. Recurrent clinical effects observed in each group, control group included, were hair loss, fur staining, sores/lesions (head, neck, ears). In addition, animals from the medium (22.3 mg/kg bw) and high (45.3 mg/kg bw) dose groups showed excessive salivation, respectively 5 and 17 animals, and three animals dosed at 45.3 mg/kg bw were thin and showed piloerection and hunched posture from day 15 of gestation. Necropsy revealed gastro intestinal lesions in one female from the control group and in four in both the medium dose group and the high dose group. Lesions were considered as minor (thickening of the mucosa surface of the stomach or duodenum), except for two out of the three animals affected by severe clinical signs from the high dose group, where gastro intestinal lesions were more severe (duodenal ulcer/abnormal ingesta). Bodyweight gain and food consumption were reduced by 4 to 16%, compared with control animals, or 3 to 6% if data from the three severely affected females were discounted.

 

- Teratogenic / Embryonic effects

All the parameters observed in the treated groups (pregnancy rate, implantation loss, number of living foetus, sex ratio…) were in the same order than those recorded in the control group, and were within the range of the historical findings. However, in the high dose group (45.3 mg/kg bw) mean foetal weight was lower than control; this is entirely attributable to the three females which showed severe clinical signs and weight loss from day 15 to 20 of gestation. If data from these females are excluded, the high dose mean foetal weight is similar to that recorded for control foetuses. Foetuses with malformations were observed in the low dose group (11.3 mg/kg bw) and in the high dose group. In the low dose group, two foetuses from two different dams, exhibited hydrocephaly or hydronephrosis. For the high dose group, one foetus showed a haemorrhagic eye and the second anophtalmia, eye socket reduced in size, cervical vertebral arches fused. This latest foetus came from one of the three females severely affected. These malformations have been observed in historical control litters, and part of them is known to occur spontaneously in this strain of rat (hydronephrosis). There was a statistical significant increase in the number of foetuses showing extra thoraco-lumbar ribs in the high dose group. The number of affected litters was also higher in this dose group than in the control group, respectively 14 and 6 litters. Supernumerary ribs occur spontaneously in most strains of rats and their frequency can be increased by many extrinsic agents.

Under the test conditions of this study, the LOAEL (maternal toxic effects) was 60 mg/kg bw/ day expressed as active substance or 45.3 mg/kg bw/day expressed as main ingredient, the NOAEL (maternal toxic effects) was 30 mg/kg bw/day expressed as active substance or 22.7 mg/kg bw/day expressed as main ingredient. The NOAEL (embryotoxic/teratogenic effects) was 60 mg/kg bw/day expressed as active substance or 45.3 mg/kg bw/day expressed as main ingredient.