Diquat dibromide

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 Apr 1988 to 03 Jun 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1989

Report date:

1989

Reference 3

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Reference 4

Reference Type:

study report

Title:

Unnamed

Year:

1995

Report date:

1995

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Version / remarks:

1998

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Diquat dibromide

EC Number:

201-579-4

EC Name:

Diquat dibromide

Cas Number:

85-00-7

Molecular formula:

C12H12N2.2Br

IUPAC Name:

1,1'-ethylene 2,2'-bipyridyldiylium dibromide

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

water

Test material form:

liquid

Test animals

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: not reported
- Weight at study initiation: 3.11 - 4.03 kg
- Housing: Individually housed in mobile rabbit units
- Diet: CRB pellets, ad libitum
- Water: Drinking water, ad libitum
- Acclimation period: 6 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 26
- Humidity (%): 46 - 66
- Photoperiod (hrs dark / hrs light): 12/12

IN LIFE DATES: From 25 Apr 1988 To: 03 Jun 1988

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Deionised water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Test substance was formulated in deionised water. The concentration of substance was adjusted to give a constant volume of 1 mL/kg body weight for each dose level. Aliquots of the dosing preparation solutions were stored at ambient conditions and fresh bottles were used for each day of the study. All animals were dosed once daily, by gavage, from days 7 - 19 inclusive of gestation with 1 mL of dosing formulation per kg body weight. The volume given to each animal was adjusted daily according to body weight. Control animals received the appropriate volume of deionised water. All animals receiving the same dose level were dosed sequentially but the order in which each group was dosed was rotated on a daily basis. Dosing was performed in the morning of each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each bulk preparation was analysed prior to the start of dosing to verify the achieved concentration of substance in deionised water. Analysis was by dilution with water followed by HPLC.

Details on mating procedure:

Animals of proven fertility were used for this study. Semen was collected from each male using an artificial vagina. The volume of the sample obtained was measured and then diluted with sufficient physiological saline to inseminate one replicate. One mL of the diluted semen was inseminated into each female using a plastic catheter. Each replicate of females was inseminated with semen from one male. After insemination, each female was given an intravenous injection of 25 IU PROFASI 500 to promote ovulation. Any individual male inseminated a maximum of 16 females (4 replicates). The day of insemination was designated a day 1 of gestation.

Duration of treatment / exposure:

From Day 7 to Day 19 (inclusive) of gestation

Frequency of treatment:

Daily

Duration of test:

Until Day 30 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: mg test substance cation/kg bw/day

Remarks:

Low dose. Group 2

Doses / concentrations 2

Dose / conc.:

3 other: mg test substance cation/kg bw/day

Remarks:

Mid dose. Group 3

Doses / concentrations 3

Dose / conc.:

10 other: mg test substance cation/kg bw/day

Remarks:

High dose. Group 4

No. of animals per sex per dose:

20

Control animals:

yes, concurrent vehicle

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS
All animals were checked on arrival to ensure that they were physically normal externally and were subsequently checked daily.

BODY WEIGHT
The body weight of each animal was recorded on Days 1 and 4, 7 - 19 (inclusive) and on Days 22, 26 and 30 of gestation.

FOOD CONSUMPTION:
The amount of food consumed by each animal was measured by giving a weighed quantity of food on Days 1, 4, 7, 10, 13, 16, 19, 22 and 26 and calculating the amount consumed from the amount left on Days 4, 7, 10, 13, 16, 19, 22, 26 and 30 respectively.

POST-MORTEM EXAMINATIONS:
- Animals which aborted or were considered to be in extremis were killed by an intravenous injection of pentobarbitone sodium solution and given an examination post mortem. Pregnancy status was recorded.
- On Day 30 of gestation the remaining animals were killed by an intravenous injection of pentobarbitone sodium solution. An examination post mortem was performed on all animals.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
The intact gravid uterus (minus ovaries and trimmed free of connective tissue) was removed and weighed. The ovaries and uterus were then examined and the following data were recorded:
(1) Number of corpora lutea in each ovary.
(2) Number and position of implantations subdivided into: live foetuses, early intra-uterine deaths and late intra-uterine deaths.

Intra-uterine deaths were classified as early or late intra-uterine deaths.
The implantations were assigned letters of the alphabet to identify their position in utero starting at the ovarian end of the left horn and ending at the ovarian end of the right horn.

Fetal examinations:

External examinations: Yes: Each foetus was examined for external abnormalities and cleft palate.
Soft tissue examinations: Yes: All foetuses were then examined internally for visceral abnormalities, sexed, eviscerated and fixed in methanol. After approximately 24 hours the head of each foetus was cut along the fronto-parietal suture line and the brain was examined for macroscopic abnormalities
Skeletal examinations: Yes: The carcasses were then returned to methanol for subsequent processing and staining with Alizarin Red S. The stained foetal skeletons were examined for abnormalities and the degree of ossification was assessed. The individual bones of the manus and pes were assessed and the result converted to a five point scale. Abnormalities were classified as major (rare or possibly lethal or both) or minor (deviations from normal that are not uncommon at external visceral or skeletal examination) defects. Variations were also recorded and classified as minor defects or variants depending on the frequency of occurrence in relation to background data
Head examinations: Yes.

Statistics:

Data relating to animals which were killed in extremis, were non-pregnant or aborted were excluded from the statistical analysis. The following data were considered by analysis of variance:
- Maternal body weight gain
- Maternal food consumption
- The numbers of implantations and live foetuses per female.
- Percentage pre-implantation loss and percentage post-implantation loss

The percentage pre-implantation loss and post-implantation loss were transformed before analysis using the double arcsine transformation of Freeman and Tukey (1950). The analyses of variance were weighted by the denominator in the proportion.

The percentage of implantations which were early intra-uterine deaths and the percentage of implantations which were late intra-uterine deaths (both calculated on an individual litter basis). The percentages were transformed before analysis using the double arcsine transformation and the analysis of variance was weighted by the number of implantations in each litter.
Gravid uterus weight, litter weight and mean foetal weight (calculated on an individual litter basis). The analysis of mean foetal weight was weighted by the number of foetuses in each litter.
Mean manus and pes score per foetus (calculated on an individual litter basis). The analyses were weighted by the number of foetuses examined in each litter. The analyses were weighted by the number of foetuses in each litter.
The percentage of foetuses with minor external/visceral defects only, and minor skeletal defects only (calculated on an individual litter basis). The percentages were transformed before analysis using the double arcsine transformation and the analyses were weighted by the number of foetuses examined in each litter.
The analyses of variance were carried out using the GLM procedure in SAS (1985). Individual treatment group means were compared with the control group mean using Student's t-test based on the error mean square in the analysis.

Indices:

Pre-implantation loss (%) = (Number of corpora lutea - Number of implantations / Number of corpora lutea) x 100
Post-implantation loss (%) = (Number of implantations - Number of live foetuses / Number of implantations) x 100

Historical control data:

In addition to historical control data presented in the original report, a supplement to the report contained further information on the variant and minor defect historical control data, together with historical control data for each of the major defects seen in this study.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no treatment-related changes in the 1 or 3 mg/kg bw/day groups. In the 10 mg/kg bw/day group there were increased numbers of animals with diarrhoea/signs of diarrhoea (although no increase in the numbers of observations), a small increase in the numbers of animals recorded as subdued, a marked increase in the incidence of thinness and a number of observations of mucus, blood and little or no faeces on the tray papers

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Six animals were killed before their scheduled termination dates. One animal from the 1 mg/kg bw/day group was killed on Day 27 following an abortion. Two 10 mg/kg bw/day animals were killed following abortion on Day 20 or Day 29. Three further animals from the 10 mg/kg bw/day group were killed in extremis on Day 17, 21 or 22.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Two 10 mg/kg bw/day females lost weight throughout dosing and then rapidly gained weight during the post-dosing period. Analyses were done both with and without these two females.
There was a clear weight loss in both the 3 mg and 10 mg/kg bw/day dose groups during the first three days of dosing. Subsequent to Day 10, weight loss (or reduced weight gain) was apparent only in certain females in the 10 mg/kg bw/day dose group (most noticeable in the same two animals).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

There was a clear, consistent and dose-related reduction in food consumption in both the 3 mg and 10 mg/kg bw/day dose groups during the first three days of dosing. For the remainder of the dosing period, mean food consumption was lower in the 10 mg/kg bw/day dose group but this was only apparent in certain females. In the 3 mg//kg bw/day dose group, certain females ate more food than others in the group during the latter half of the dosing period.
Post-dosing, some females in both the 3 mg and 10 mg/kg bw/day dose groups ate more food with consequently higher mean food consumption. The increases in food consumption during the latter period (3 mg and 10 mg/kg bw/day groups) may indicate a compensatory effect for the earlier reductions.

Food efficiency:

not examined

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

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The major findings were changes in the alimentary tract which were seen most commonly in the intercurrent deaths in the 10 mg/kg bw/day group. Of these, the most important change was haemorrhage and/or ulceration in the stomach which was also seen in two animals (one each in the 1 and 10 mg/kg bw/day groups) surviving to termination. Prominent reticular pattern in the liver was also seen in a number of animals, particularly in the intercurrent deaths. One unusual finding in the intercurrent deaths was changes in the intestinal blood vessels; in one animal, the vessels in the caecum appeared to have leaked.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Maternal developmental toxicity

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

Three animals aborted, one in the 1 and two in the 10 mg/kg bw/day groups respectively.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

not examined

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: test substance cation species

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Original value presented in study

2

Key result

Dose descriptor:

NOAEL

Remarks:

Maternal toxicity

Effect level:

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

Based on:

other: pure test substance

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

effects observed, non-treatment-related

Reduction in number of live offspring:

effects observed, non-treatment-related

Changes in sex ratio:

effects observed, non-treatment-related

Changes in litter size and weights:

effects observed, non-treatment-related

Changes in postnatal survival:

effects observed, non-treatment-related

External malformations:

no effects observed

Description (incidence and severity):

The overall incidence of external defects was unaffected by treatment with test substance.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

Overall, there was a dose-related increase in the percentage of foetuses with minor skeletal defects. The difference between the control group and the 3 mg and 10 mg/kg bw/day dose groups attained statistical significance using a foetus-based analysis (Fisher's Exact Test). However, the difference between the control group and the 1 mg/kg bw/day dose group was marginal and not statistically significant. Using a litter-based analysis (double arcsine transformation and analysis of variance) which is considered to be more appropriate for defect data, there were no statistically significant differences compared with the control group for any of the test groups.
Specific minor defects to show increased statistical incidence in any dose group were:
- Not ossified 6th sternebra, and
- partially ossified 6th sternebra.
Both these defects were statistically significantly increased at the 10 mg/kg bw/day dose level. Partially ossified 6th sternebra was also higher than control at the 3 mg substance /kg/day dose level. A re-examination of the ventral tubercle was carried out because the incidence reported was found to be inconsistent with historical data, i.e. no partially ossified ventral tubercles from control foetuses have been seen either before or after this study. It was the opinion of the re-evaluator that all of the foetuses examined exhibited ventral tubercles which were within normal limits and all are considered to be fully ossified. Therefore no ventral tubercles in this study are considered to be partially ossified following this re-evaluation.
Not ossified 6th sternebra was statistically significantly increased at the 1 mg/kg bw/day dose level but the frequency was lower at 3 mg than 1 mg/kg bw/day and hence there was no dose response at least at the two lower levels. These two defects in combination showed an apparent dose response, but all values were within historical control values. The control value for the present study appears to be unusually low. Only three foetuses did not have a skeletal variant. Only three foetuses did not have a skeletal variant.
The incidence of foetuses with major defects was 2, 8, 2 and 4 in each dose group respectively. Since most specific major defects occurred as single incidences in one (or two) treatment groups they were considered to be spontaneous occurrences and not as a result of treatment with substance.
There was a clear increase in the proportion of foetuses with pes score 2 in the 10 mg/kg bw/day dose group. There was a similar increase in the proportion of foetuses with manus score 2, also in the 10 mg substance /kg/day dose group. This did not achieve statistical significance although was reflected in the mean score. There was no evidence of an effect at the two lower doses.
There was a statistically significant increase in the incidence of 27 pre-sacral vertebrae in the 10 mg/kg bw/day dose group although this was at the upper end of the control range. There was an increased incidence of 13th extra ribs in both the 3 mg and 10 mg/kg bw/day dose group. Extra 13th ribs showed a similar increase in incidence to that of 27 pre-sacral vertebrae and again the incidence at 10 mg/kg bw/day was at the upper end of the control range.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The overall incidence of external/visceral defects was unaffected by treatment with test substance. Three specific minor external/visceral defects observed were:
- Friable liver which was increased in the 10 mg/kg bw/day group.
- Mottled liver which was increased in the 10 mg/kg bw/day group and tended to occur in the foetuses with friable liver.
- Gall bladder: small blood clots attached - the incidence of foetuses with this defect in the 3 mg/kg bw/day dose group was well above the historical control range but there was clearly no dose response.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: sternebra ossification

Remarks on result:

other:

Remarks:

Original value presented in study

2

Key result

Dose descriptor:

NOAEL

Remarks:

Developmental toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: sternebra ossification

Remarks on result:

other:

Remarks:

Recalculated value, expressed as pure substance, see ‘Any other information on results incl. tables’ for respective calculation

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: sternum

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

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

Treatment related:

yes

Relation to maternal toxicity:

not specified

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Diet/dosage preparation and analysis

- Concentration analysis: Mean test substance concentrations were within 7 % of the nominal values.

- Homogeneity: The preparations formed solutions in deionised water.

- Stability: The chemical stability of test substance in deionised water after an interval of 23 days (which is the same as the dosing period in the present study) was assessed in a preliminary study in the rabbit and found to be satisfactory.

 

Calculation of key result

The original effect levels were expressed as cation species of the test substance. The key effect levels are re-calculated and corrected to include the counterion species by multiplying with 1.868 (344.0 g/mol molecular weight of registered substance divided by 184.2 g/mol molecular weight of cation species).:

The NOAEL for maternal toxicity = 1.868 x 1 mg/kg bw/day = 1.9 mg/kg bw/day

The NOAEL for fetal developmental toxicity = 1.868 x 3 mg/kg bw/day = 5.6 mg/kg bw/day

 

Table 1: Intergroup comparison of body weight gain (g) all females

	Dose Level of test substance (mg/kg/day)
Period (days)	0 (control)	1	3	10 (all females)	10 mg/kg/day Excluding 2 females
Initial wt (1)	3418.1	3401.3	3344.5	3368.2	3357.1
Pre-dosing (1 – 7)	130.2	131.0	158.4	171.9	165.0
During dosing (7 – 19)	237.4	309.9	236.4	47.5**	175.1
Post dosing (19 – 30)	312.4	387.1	293.3	424.8*	352.4
Overall (1 – 30)	680.1	828.0*	688.2	644.3	692.5

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 2: Intergroup comparison of food consumption (g/day)

	Dose Level of test substance (mg/kg/day)
Period (days)	0 (control)	1	3	10
Pre-dosing (1 – 7)	173.2	162.4	168.8	172.3
During dosing (7 – 19)	163.4	158.1	150.1	96.0**
Post dosing (16 – 30)	164.1	170.4	181.0*	186.6*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 3: Caesarean section observations for all pregnant females

Observation	Dose Level of test substance (mg/kg/day)
	0 (control)	1	3	10
Animals inseminated	20	20	20	20
Animals Pregnant	18	16	20	17
Number of females that aborted	0	1	0	2
Number of intercurrent deaths (including females with abortions)	0	0	0	3^^
Number of females with live foetusesin uteroat termination	18	15	20	13
Corpora Lutea/Dam	11.50	11.93	9.35	10.46
Pre-implantation Loss (%)	16.4	12.3	18.2	14.7
Number of females affected	11/18	9/15	12/20	5/13
Mean number of implantations	9.61	10.47	7.65*	8.92
Post-implantation Loss (%)	15.0	14.6	15.7	17.2
Number of females affected	13/18	9/15	14/20	8/13
Mean number of live foetuses	8.17	8.93	6.45*	7.38
Number of intra-uterine deaths %                  Early                  Late	6.9 8.1	3.8 10.8	6.5 9.2	9.5 7.8
Intra-uterine deaths/Dam
Early (Proportion of litters affected)	8/18	4/15	7/20	5/13
Late (Proportion of litters affected)	6/18	7/15	8/20	5/13
Total number of live foetuses	147	134	129	96
Sex Ratio (% Males per litter)	54.4	49.6	49.6	47.9
Mean gravid uterus weight (g)	483.1	539.7	394.9*	413.3
Mean Litter Weight (g)	325.1	344.8	271.4	273.6
Mean Foetal Weight (g)	39.81	38.59	42.08	37.05

^ Including one animal that was not pregnant.

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

 

Table 4 Intergroup comparison of selected foetal defects and variants

Observation	Dose Level of test substance (mg/kg/day)
	0 (control)	1	3	10
No. of litters examined	18	15	20	13
No. of foetuses examined	147	134	129	96
Total incidence of major defects	2	8	2	4
No. of foetuses showing major external/visceral defects	2	8*	2	3
No. of foetuses showing major skeletal defects	1	3	1	1
No. of foetuses showing minor external/visceral defects only	60	56	64	39
No. of foetuses showing minor skeletal defects only	64	65	71*	53*
Liver – friable (foetus/litter)	8/5	7/2	5/2	14*/3
Gall bladder – small blood clot(s) attached (foetus/litter)	18/8	21/9	33**/13	14/8
Vertebral column – 27 pre-sacral vertebrae (foetus/litter)	46/14	54/12	49/15	49**/12
Sternebrae – not ossified 6^th(foetus/litter)	0/0	6*/2	3/3	6**/5**
Sternebrae – partially ossified 6^th(foetus/litter)	9/6	14/8	19*/8	16**/8
Extra ribs – 13^thnormal length (foetus/litter)	76/15	61/13	89**/18	71**/12
Mean manus score	1.61	1.60	1.62	1.80
Mean pes score	1.43	1.45	1.40	1.65*

* Statistically significant difference from control group mean, p<0.05 (Student’s t-test, 2-sided)

** Statistically significant difference from control group mean, p<0.01 (Student’s t-test, 2-sided)

Applicant's summary and conclusion

Conclusions:

Test substance administered to rabbits by gavage during gestation at a concentration of 10 mg test substance ion/kg bw/day, equivalent to 19 mg pure test substance/kg bw/day, caused maternal toxicity and foetotoxicity. Mild maternal toxicity was seen with 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day. The NOAEL for maternal toxicity was set at 1 mg test substance ion/kg bw/day, corresponding to 1.9 mg pure test substance/kg bw/day. A dose of 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day, was considered to be a no-effect level for embryonic and foetal development.

Executive summary:

A study, complying to GLP and OECD guideline 414, investigated the effect of the test substance when administered by gavage during the period of organogenesis on the embryonic and foetal development of the rabbit. Groups of 20 young adult female New Zealand White rabbits were dosed by gavage with 1, 3 or 10 mg test substance cation/kg bw/day in deionised water from Days 7 - 19 (inclusive) of gestation which thus included the period of major organogenesis. A control group of animals received deionised water alone. The day of insemination was designated Day 1 of gestation. On Day 30 of gestation the females were killed and their uteri examined for live foetuses and intra-uterine deaths. The foetuses were weighed, killed, examined for external and visceral abnormalities, sexed, eviscerated and stained for skeletal examination.

Administration of 10 mg/kg bw/day resulted in maternal toxicity which was apparent as weight loss and reduced food consumption. Five animals (including two which aborted) from this group were killed prematurely. Similar but less severe effects on body weight and food consumption were seen at 3 mg/kg bw/day. There were no treatment-related effects on numbers of corpora lutea, pre-or post-implantation loss, litter size, sex distribution, gravid uterus weight or litter weight. Foetal weight was marginally lower at 10 mg/kg bw/day. Sixteen foetuses were seen with major abnormalities (2, 8, 2 and 4 foetuses in the control, 1, 3 and 10 mg/kg bw/day groups, respectively). There was no evidence that the type or distribution of these was related to treatment. The overall incidence of foetuses with minor external/visceral defects was not affected by treatment although the incidence of mottled and friable livers was increased at 10 mg/kg bw/day. There was a small increase in the percentage of foetuses with minor skeletal defects at 3 and 10 mg test substance/kg/day. The incidences were not statistically significant using a litter-based method of analysis. Specifically, there were increased numbers of partially ossified 6th sternebra at both levels while not ossified 6th sternebra showed an increase at 10 mg/kg bw/day only. A re-examination of the ventral tubercle was carried out because the incidence reported was found to be inconsistent with historical data, i.e. no partially ossified ventral tubercles from control foetuses have been seen either before or after this study. It was the opinion of the re-evaluator that all of the foetuses examined exhibited ventral tubercles which were within normal limits and all are considered to be fully ossified. Therefore no ventral tubercles in this study are considered to be partially ossified following this re-evaluation. The frequency of the skeletal variant 27 pre-sacral vertebrae was increased at 10 mg/kg bw/day while the incidence of extra thoracic ribs was increased at both 3 and 10 mg/kg bw/day although in all cases these were within recent historical control incidences. Manus and pes scores were increased at 10 mg test substance cation/kg/day. These scores, which are generally sensitive indicators of reduced ossification, were similar in the 1 and 3 mg/kg bw/day groups to those of the controls.

Test substance administered to rabbits by gavage during gestation at 10 mg test substance cation/kg bw/day, corresponding to 19 mg pure test substance/kg bw/day, caused maternal toxicity and foetotoxicity. Mild maternal toxicity was seen at 3 mg test substance cation/kg bw/day, equivalent to 5.64 mg pure test substance/kg bw/day. The NOAEL for maternal toxicity was determined to be 1.9 mg pure test substance /kg bw/day.  The dose of 3 mg test substance ion/kg bw/day, equivalent to 5.64 mg pure test substance salt/kg bw/day, was considered to be the NOAEL for embryonic and foetal development.