Paraquat-dichloride

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

3 Jun 1992 to 19 Jun 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Paraquat-dichloride

EC Number:

217-615-7

EC Name:

Paraquat-dichloride

Cas Number:

1910-42-5

Molecular formula:

C12H14N2.2Cl

IUPAC Name:

1,1’-dimethyl-4,4’-bipyridyldiylium dichloride

Test material form:

liquid

Test animals

Species:

mouse

Strain:

CD-1

Remarks:

Crl:CD-1(ICR)BR

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 8 weeks.
- Weight at study initiation: 25 to 29 g.
- Housing: During acclimatisation and mating the animals were housed in grid bottomed polypropylene cages over paper-lined trays. Males were housed individually and females in groups of up to five, except when paired for mating. Mated females were individually housed in solid bottomed polypropylene cages, with wood sawdust provided as bedding.
- Diet: Pelleted rodent diet, ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: 11 days prior to mating.

DIET AND WATER QUALITY VERIFICATION
- Each batch of diet was delivered with an accompanying certificate of analysis detailing nutritional composition and levels of specified contaminants (heavy metals, aflatoxins and insecticides). The water is periodically analysed by independent analysts for microbiological purity and levels of halogenated hydrocarbons. There were no known contaminants in the food or water that were considered likely to interfere with the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 to 25
- Humidity (%): 39 to 68
- Air changes (per hr): 16
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES:
3 Jun 1992 to 19 Jun 1992

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

purified

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Dosing solutions were prepared once for the study, prior to the start off dosing, and divided into appropriate aliquot portions. Dosing solutions were prepared with purified water. The weighed quantity of test material was dissolved in the appropriate volume of vehicle. Separate solutions were prepared, corrected for purity content, for each dose level. The aliquots were stored at room temperature protected from light, until required for dosing.

VEHICLE
- Amount of vehicle: 10 mL/kg bw calculated and adjusted daily on an individual body weight basis.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

As a check on the accuracy of preparation, samples from each dosing solution were determined by high performance liquid chromatography (HPLC) with ultraviolet (UV) detection. The method of analysis was validated in a previous study.

Details on mating procedure:

- Impregnation procedure: cohoused.
- M/F ratio per cage: 1/1.
- Length of cohabitation: Overnight on four consecutive days.
- Proof of pregnancy: vaginal plug (either in situ or in the cage tray) referred to as day 0 of pregnancy.

Duration of treatment / exposure:

Days 6 to 15 of pregnancy inclusive.

Frequency of treatment:

Daily.

Duration of test:

Till day 18 of pregnancy.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 2, low dose group. Concentration of test substance cation.

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 3, mid dose group. Concentration of test substance cation.

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 4, high dose group. Concentration of test substance cation.

No. of animals per sex per dose:

26

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected by the Sponsor, on the basis of results from a preliminary study performed.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily from day 0 of pregnancy.

BODY WEIGHT: Yes
- Time schedule for examinations: Days 0, 6 to 15, inclusive and 18 of pregnancy. The dead body weight of the killed animals was recorded.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule: Days 0 to 6, 6 to 9, 9 to 12, 12 to 15 and 15 to 18 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on day 18 of pregnancy.
- Organs examined: major organs, lungs plus trachea, and kidneys were removed and weighed.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: No
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: number and distribution of implantations in uterine horns classified as early resorptions/late resorptions/dead fetuses/live fetuses.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter (half fixed in Bouin’s fluid, the rest in 70% alcohol)
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

The following parameters were analysed by analysis of variance: maternal body weight, maternal body weight gain, maternal food consumption, maternal organ weights, maternal body weight-related organ weights, the numbers of implantations and live fetuses per female, percentage post-implantation loss, percentage of male fetuses, litter weight and mean fetal weight.
Fetal data was analysed on litter basis. Percentages were transformed before analysis using the double arcsine transformation.
Each treatment group was compared to the control group using Student's t-test, based on the error mean square from the analysis of variance. The tests were two-sided and statistical significance was set at p < 0.05 and 0.01.
Fisher's exact probability test was used to compare each treated group with the control group for the following: the proportion of foetuses with each individual defect, the proportion of litters with each individual defect
The tests were one-sided and statistical significance was set at p < 0.05 and 0.01 .

Indices:

percentage post implantation loss and percentage of male foetuses.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The animals which were terminated prematurely showed prior to death piloerection, laboured respiration, hunched posture, were hypothermic, hypoactive and/or had pale extremities and eyes. There were no other premature deaths and no other treatment-related changes in clinical conditions were observed in other treatment groups.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

In the group treated at 25 mg/kg bw/day, one female was found dead on day 16 of pregnancy. There were no changes in clinical condition observed prior to death. Four other females in this dose group were terminated prematurely, due to poor clinical condition at the end of the dosing period/beginning of the post dosing period (days 16, 16, 17 and 15 of pregnancy, respectively).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There was an effect of treatment at 25 mg/kg bw/day on maternal body weight gain. A statistically significant (p < 0.01) retardation of maternal body weight gain over days 12 to 15 and 15 to 18, and also over the entire dosing period (days 6 to 15) and the entire pregnancy period (days 0 to 18) was seen in comparison with the control group. Body weight gain of this group from day 0 to day 18 after adjustment for gravid uterus weight was also slightly retarded in comparison with the control group, but statistical significance was not achieved. Body weight itself in this group was significantly lower than the control group on day 15 (p < 0.01) and day 18 (p < 0.01) of pregnancy (Table 1 and 2 in 'Any other information on results incl. tables').
At 7.5 or 15 mg/kg bw/day, maternal body weight gain was unaffected by treatment and body weights and adjusted body weight gains were similar to the control group, throughout pregnancy.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

At 25 mg/kg bw/day, food consumption from days 12 to 15 of pregnancy was reduced in comparison with the control group. However, although there was statistically significant (p < 0.05) variation amongst the groups, the difference between the group treated at 25 mg/kg bw/day and the control group was not statistically significant. Food consumption from days 15 to 18 of pregnancy in this group was slightly lower than the control group, but the difference was not statistically significant.
There was no effect of treatment at 7.5 or 15 mg/kg bw/day on maternal food consumption. There was intergroup variation throughout pregnancy, but the females in these two treated groups consumed a similar or slightly greater amount of food than the control group.

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:

effects observed, treatment-related

Description (incidence and severity):

There was an effect of treatment at 25 mg/kg bw/day on lung with trachea and kidney weights (Table 3 in 'Any other information on results incl. tables'). For all females (including pregnant, not pregnant and those terminated prematurely or found dead) treated at 25 mg/kg bw/day, although mean dead body weight was significantly (p < 0.01) lower than the control group, the mean absolute and body weight-related lung with trachea weights were significantly (p < 0.01) greater than the control group. Body weight-related kidney weight of this group was also slightly, but not statistically significantly, greater than the control group, although absolute kidney weight was slightly lower. For pregnant females only, killed on day 18, treated at 25 mg/kg bw/day, the same pattern of increased body weight-related lung with trachea and kidney weights were observed but the differences were not as great as for all females.
There was no effect of treatment at 7.5 or 15 mg/kg bw/day on lung with trachea or kidney weights. Although there was an apparent slight dose-related increase in body weight-related lung with trachea weights for all females (including pregnant, not pregnant and those terminated prematurely) treated at 7.5 or 15 mg/kg bw/day, this was not observed for pregnant females only, killed on day 18. The apparent increase was because there were more non-pregnant females in these treated groups and with their lower body weights, their organs were slightly heavier in comparison. Absolute and body weight-related kidney weights in these two groups were similar to those of the control group.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Of the five females in the group treated at 25 mg/kg bw/day, that were killed prematurely or found dead, three had no food present in the stomach, the one female found dead had stomach and intestines distended with gas and all five had dark red lung lobes. All five were pregnant and had live foetuses in utero at the time of maternal death. Dark red lung lobes were observed at the scheduled necropsy on day 18 of pregnancy for four other females in this group. No other treatment-related abnormalities were observed for this group. There were no treatment-related abnormalities observed in any of the females from the groups treated at 7.5 or 15 mg/kg bw/day.

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:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Mean post-implantation loss was similar in all groups.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There was no effect of treatment at any of the dose levels investigated on the number of live fetuses. At 25 mg/kg bw/day, the mean numbers of implantations and live foetuses were slightly, but not statistically significantly, lower than the control group (Table 4 in 'Any other information on results incl. tables'). However, values were within historical control ranges and therefore the changes were considered not to be biologically significant. The five females in the 25 mg/kg bw/day treatment group that were killed prematurely or found dead had live foetuses in utero at the time of maternal death.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There were 24, 21, 22 and 19 females pregnant in the control group and groups treated at 7.5, 15 or 25 mg/kg bw/day, respectively (Table 4 in 'Any other information on results incl. tables').

Other effects:

not examined

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: test material cation

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

original value presented in study.

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: pure test substance

Basis for effect level:

body weight and weight gain

clinical signs

gross pathology

mortality

organ weights and organ / body weight ratios

Remarks on result:

other:

Remarks:

recalculated value, expressed as pure test 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, treatment-related

Description (incidence and severity):

There was an effect of treatment at 25 mg/kg bw/day on fetal body weight (Table 5 in 'Any other information on results incl. tables'). Mean fetal weights were significantly lower than in the control group (p < 0.01 male fetuses and all fetuses, p < 0.01- female fetuses) and, consequently, mean gravid uterus weight was significantly (p < 0.01) lower than the control group.
At 7.5 or 15 mg/kg/day, fetal weights and gravid uterus weights were slightly, but not statistically significantly, lower than the control group. However, mean values were within historical control ranges and therefore the changes were considered not to be biologically significant.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of treatment on the sex ratio of fetuses with near equal proportions of male and female fetuses in each group (Table 4 in 'Any other information on results incl. tables').

Changes in litter size and weights:

effects observed, treatment-related

Description (incidence and severity):

Since fetal body weights in the 25 mg/kg bw/day dose group were significantly lower than in the control group, mean gravid uterus weight was significantly (p < 0.01) lower than the control group (Table 5 in 'Any other information on results incl. tables'). At 7.5 or 15 mg/kg bw/day, fetal weights and therefore gravid uterus weights were slightly, but not statistically significantly, lower than the control group. However, mean values were within historical control ranges and therefore the changes were considered not to be biologically significant.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

MAJOR ABNORMALITIES
No external major abnormalities without skeletal origin were observed.

MINOR ABNORMALITIES
There was no dose-related trend and values were within historical control range, there was considered to be no effect of treatment on the incidence of minor external abnormalities.

Skeletal malformations:

effects observed, treatment-related

Description (incidence and severity):

MAJOR ABNORMALITIES
The incidence of fetuses or litters with major abnormalities did not show a treatment-related increase. One fetus in the group treated at 7.5 mg/kg bw/day showed spina bifida and in the group treated at 15 mg/kg bw/day, one showed cleft palate. These abnormalities were considered to be spontaneous and unrelated to treatment.

MINOR ABNORMALITIES
There was no statistically significant or dose-related increase in the numbers of fetuses or litters with minor skeletal abnormalities or in the numbers of fetuses or litters with any particular type of minor skeletal abnormalities. All incidences were within historical control ranges .

VARIATIONS
There was a statistically significant (p < 0.01) increase in the numbers of fetuses in the 25 mg/kg bw/day group with six or less caudal centra, and astragalus not ossified. There was a slight, but not statistically significant, increase in the incidence of fetuses with three or less caudal neural arches. The incidences of these findings in this group were above historical control ranges. There was also a slight, but not statistically significant, increase in the numbers of fetuses in the 25 mg/kg bw/day group with retarded ossification of the occipital and with one or more sternebrae bilobed, bipartite, misaligned or mis-shapen.
The incidences of these findings were with historical control ranges. There were significantly (p < 0.05) greater numbers of litters in the 25 mg/kg bw/day group affected with retarded ossification of the occipital, astragalus not ossified and six or less caudal centra. These findings were considered to be as a result of the lower fetal weights in the 25 mg/kg bw/day group. The incidence of fetuses with the variant of six or less caudal centra ossified was unusually low in the control group and, in comparison, the incidences in the groups treated at 7.5 or 15 mg/kg bw/day appeared high. However the values in these treated groups were within the historical control range and were close to the historical mean so it was concluded that there was no association with treatment.
There was a higher number of fetuses from all treated groups with 14 thoracic vertebrae and five lumbar vertebrae in comparison with the control group. The numbers of fetuses affected were significantly (p < 0.05) different from the control group in the 15 and 25 mg/kg bw/day groups, for both findings but the numbers of litters affected did not show statistically significant or dose-related increases. There were also higher numbers of fetuses with vestigial and/or extra 14th ribs. The numbers of fetuses affected was significantly greater than the control group for unilateral or bilateral vestigial 14th (p < 0.01 7.5 mg/kg bw/day, p < 0.05 25 mg/kg bw/day) and unilateral or bilateral extra 14th rib (p < 0.05 - 15 or 25 mg/kg bw/day). Consequently, there was a lower number of fetuses and litters in the treated groups with 13 normal pairs of ribs and, for numbers of fetuses, the difference from the control group was significant (p < 0.01) for all treated groups.
These five variants are all related. The fetuses show the same numbers of vertebrae but the presence of extra or vestigial ribs classified the vertebra at the thoraco-lumbar border as thoracic instead of lumbar; hence the shift in the proportions. The thoraco-lumbar border is an extremely labile region and so fluctuations in incidences are not unexpected. As a consequence of this, and also since the incidences of these variants were within or only slightly above historical control ranges and no dose-related trends were observed, the findings were considered to be fortuitous and unrelated to treatment.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

MAJOR ABNORMALITIES
The incidence of fetuses or litters with major abnormalities did not show a treatment-related increase. One fetus in the group treated at 15 mg/kg bw/day showed kidney agenesis. This abnormality was considered to be spontaneous and unrelated to treatment.

MINOR ABNORMALITIES
There was a significantly higher number of fetuses with minor external/visceral abnormalities in the 7.5 and 15 mg/kg bw/day groups (p < 0.01 and p < 0.05, respectively) and a significantly (p < 0.01) higher number of litters affected in the 7.5 mg/kg bw/day group in comparison with the control group. However, the numbers of fetuses and litters affected in the 25 mg/kg bw/day group was not statistically different from the control group. Since there was no dose-related trend and values were within historical control range, there was considered to be no effect of treatment on the incidence of minor visceral abnormalities.
The higher numbers in the 7.5 and 15 mg/kg bw/day groups were due to significantly greater numbers of fetuses with increased renal pelvic cavitation (p < 0.01 and p < 0.05, respectively). There was also a slightly greater number of fetuses with this minor abnormality in the 25 mg/kg bw/day group, in comparison with the control group. There was a similar, less pronounced, trend in the number of litters affected, but statistical significance was not observed. Although the incidence of this abnormality in the treated groups was above the historical control range, there was no dose-related trend so this finding was considered to be fortuitous and unrelated to treatment.

Other effects:

not examined

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Remarks:

original value presented in study

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

skeletal malformations

Remarks on result:

other:

Remarks:

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

Fetal abnormalities

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

skeletal: supernumerary rib

skeletal: vertebra

Description (incidence and severity):

The findings are in line with retardation of embryonic/foetal growth.

Overall developmental toxicity

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

25 other: mg/kg bw/day (actual dose received) recalculated value, expressed as pure test substance, see ‘Any other information on results incl. tables’ for respective calculation).

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Verification of dosing solution

The results of analysis of dosing solutions demonstrated that they were prepared correctly and that they were stable for up to one month, which exceeded the period of use, when stored at room temperature in the dark.

 

Calculation of key result maternal and developmental toxicity

The doses of the test substance were expressed in test substance cation, which relates to the cation species in an aqueous solution of the registered substance. The effect levels are already corrected for the amount of water. The key effect levels are calculated by inclusion of the anion species:

(100/72.4) x 15 mg test substance cation / kg bw = 20.7 mg pure pure test substance/ kg bw

(100/72.4) x 25 mg test substance cation / kg bw = 34.5 mg pure pure test substance/ kg bw

Table 1. Group maternal body weights (g) – (mean ± SD).

Day of gestation   Group	n	0	6	7	8	9	12	15	18	18#
1	24	27.9 ± 1.7	31.0 ± 1.8	32.2 ± 1.9	33.1 ±1.7	33.9 ± 1.8	39.9 ± 2.1	47.9 ± 3.0	59.0 ± 4.1 (23)	34.7 ± 2.6 (21)
2	21	28.1 ± 1.6	30.9 ± 1.6	31.9 ± 1.6	32.5 ± 1.7	33.3 ± 1.7	39.1 ± 2.3	46.8 ± 3.0	58.3 ± 4.2	34.3 ± 2.6 (19)
3	21	28.0 ± 2.0	31.0 ± 2.2	31.9 ± 2.0	32.6 ± 2.1	33.5 ± 2.2	38.9 ± 3.0	47.0 ± 3.7	58.9 ± 5.6	35.2 ± 3.2 (21)
4	19	28.4 ±1.8	31.7 ± 2.6	32.2 ± 2.1	32.9 ± 1.9	33.9 ± 1.8	39.5 ± 2.5	44.8 ± 3.5 **	53.7 ± 3.9 (14) **	33.9 ± 3.1 (13)
Analysis of variance		NS	NS	NS	NS	NS	NS	p<0.05	p<0.01	NS

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

# = adjusted for gravid uterus weight

Table 2. Group maternal body weights (g) – (mean ± SD).

Day of gestation   Group	n	0 - 6	6 - 9	9 - 12	12 - 15	15 - 18	6 - 15	0 - 18	0 - 18#
1	24	3.1 ± 1.1	2.9 ± 0.6	6.0 ± 0.8	8.0 ± 1.2	11.3 ± 1.7 (23)	16.9 ± 2.1	31.3 ± 3.4 (23)	7.1 ± 2.3 (21)
2	21	2.9 ± 0.9	2.3 ± 0.6	5.9 ± 1.4	7.7 ± 1.3	11.4 ± 1.5	15.9 ± 2.4	30.2 ± 4.1	6.4 ± 2.7 (19)
3	21	3.0 ± 0.8	2.5 ± 0.7	5.4 ± 1.2	8.1 ± 1.0	11.5 ± 2.5	16.1 ± 1.9	30.5 ± 4.2	7.2 ± 2.0 (21)
4	19	3.3 ± 1.7	2.3 ± 1.4	5.6 ± 1.3	5.3 ± 3.2**	8.0 ± 2.5** (14)	13.1 ± 3.9**	25.4 ± 3.9 ** (14)	5.8 ± 2.9 (13) **
Analysis of variance		NS	NS	NS	p<0.01	p<0.01	p<0.01	p<0.01	NS

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

# = adjusted for gravid uterus weight

Table 3. Group mean maternal absolute (g) and body weight related organ weights (g/kg) of pregnant females killed on day 18 – (mean ± SD).

			Absolute weight (g)		Body weight related (g/kg)
Group	n	Dead body weight (g)	Lungs & trachea	Kidney total	Lungs & trachea	Kidney total
1	23	57.6 ± 4.2	0.244 ± 0.032	0.528± 0.042	4.276 ± 0.591	9.191 ± 0.789
2	21	57.1 ± 4.2	0.249 ± 0.023	0.529 ± 0.043	4.508 ± 0.753	9.302 ± 0.767
3	21	57.8 ± 5.3	0.250 ± 0.034	0.527 ± 0.043	4.338 ± 0.598	9.163 ± 0.837
4	14	52.7 ± 4.1 **	0.271 ± 0.038	0.515 ± 0.075	5.155 ± 0.770 **	9.763 ± 1.102
Analysis of variance		p<0.01	NS	NS	p<0.01	NS

n = number of animals in mean data

** = significantly different from control, p<0.01 Student’s t-test

 

Table 4. Group mean pregnancy data

Group	Number of pregnant/mated	Mean no. of implantations ± SD	Mean no. of live fetuses ± SD	Mean post-implantation loss (%)	Sex ratio (%) M : F
1	24/26	13.7 ± 2.0	13.0 ± 2.3	5.5	55:45
2	21/26	13.5 ± 2.1	12.7 ± 2.5	6.2	48:52
3	22/26	13.9 ± 2.1	13.0 ± 2.3	6.3	48:52
4	19/26	12.3 ± 2.9	11.7 ± 3.0	5.0	51:49
Analysis of variance		NS	NS
Kruskal-Wallis test				NS	NS

 

 

Table 5. Group mean fetal gravid uterus weights (g) – (mean ± SD)

Group	n	Fetuses (male and female)	Uterus
1	23	1.41 ± 0.08	24.1 ± (21)
2	21	1.38 ± 0.09	23.8 ± 3.3 (19)
3	22	1.37 ± 0.08	23.7 ± 3.7 (21)
4	14	1.28 ± 0.16**	20.0 ± 2.6(13) **
Analysis of variance		p<0.01	p<0.01

n = number of animals in means

() = n where differs from original

** = significantly different from control, p<0.01 Student’s t-test

 

Applicant's summary and conclusion

Conclusions:

In this GLP compliant developmental toxicity study, performed similar to an OECD 414 test protocol, maternal NOAEL of 15 mg test substance cation/kg bw/day can be set based on clinical signs, body weight and gross pathology. The developmental NOAELs of 15 mg test substance cation/kg bw/day can be set based decreases in mean fetal weights and retarded ossification of the occipital, increases in the number with 6 caudal centra, increases in the number with uni- or bilateral extra 14th ribs and increases in the number with non-ossified astragalus in the hindlimb seen at 25 mg test substance cation/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity are 20.7 mg pure test substance/kg bw/day. The developmental effects are considered secondary to the maternal effects.

Executive summary:

In this GLP compliant developmental toxicity study, performed similar to a study according to OECD TG 414, the test substance was tested for embryonic and fetotoxic effects in Crl:CD-1 mice. The test material was administered by gavage (10 mL/kg bw) in an aqueous solution of purified water at daily doses of 7.5, 15 and 25 mg test substance cation/kg body weight from gestation day 6 to 15. The control group was dosed with purified water only. Each group consisted of 26 time-mated mice (Crl:CD-1 (ICR)BR). Maternal clinical signs, body weights and food consumption were recorded. On day 18 of gestation the females were killed. All females were subject to necropsy at which lungs with trachea and the kidneys were removed and weighed. Pregnancy status was assessed and the gravid uterus weight and numbers of live and dead implantations were recorded. Live fetuses were weighed and, after light fixation in alcohol (70%), were examined for external abnormalities and sexed. One half of the fetuses were replaced in 70% alcohol and then examined for visceral abnormalities and eviscerated before being cleared in potassium hydroxide, stained with alizarin red S and examined for skeletal abnormalities. The remaining fetuses were fixed in Bouin's fluid and were examined for visceral abnormalities.

In the 25 mg/kg bw/day group, there was an effect of treatment on maternal clinical condition. Four females were affected and were consequently terminated prematurely between days 15 and 17 of pregnancy. The effects included piloerection, laboured respiration, hunched posture, hypothermia, hypoactivity and pale extremities and eyes. One female in this group was found dead on day 16 of pregnancy, but no changes in clinical condition were observed prior to death. There was an effect of treatment on maternal body weight and food consumption. Body weight gain was retarded and food consumption was reduced, in comparison with the control group, from approximately day 12 of pregnancy until necropsy on day 18. The four females terminated prematurely, the one female found dead and four other females in this group had dark red lung lobes at necropsy. Three females also had no food present in the stomach. Lung and kidney weights were affected by treatment. Absolute and body weight-related lung with trachea weights and body weight-related kidney weights were greater than in the control group.

The numbers of implantations or live fetuses, post-implantation loss or fetal sex ratio were not affected by treatment. Retardation of embryonic/fetal growth was seen in the 25 mg/kg bw/day group. Mean fetal weight was lower than in the control group, and there was an increase in the incidence of certain variants of skeletal ossification (retarded ossification of the caudal vertebrae, occipital and astragalus, and bilobed, bipartite, misaligned and mis-shapen sternebrae). There was no evidence of teratogenicity at this dose level.

In the 7.5 and 15 mg/kg bw/day groups, there was no effect of treatment on clinical condition, body weight or food consumption of dams and there were no premature deaths. There were no treatment-related abnormalities at necropsy. Lung and kidney weights were not affected by treatment. There was no effect of treatment at this dose level on the numbers of implantations or live fetuses, post-implantation loss of fetal sex ratio. Retardation of embryonic/fetal growth was not seen in this group and there was no evidence of teratogenicity.

Maternal toxicity and developmental toxicity were seen at the high dose group (25 mg test substance cation/kg bw bw/day). The maternal NOAEL of 15 mg test substance cation/kg bw/day can be set based on clinical signs, reduced body weight and gross pathology. The developmental NOAELs of 15 mg test substance cation/kg bw/day can be set based on decreases in mean fetal weights and retarded ossification of the occipital, increases in the number with 6 caudal centra, increases in the number with uni- or bilateral extra 14th ribs and increases in the number with non-ossified astragalus in the hindlimb seen at  a dose of 25 mg test substance cation/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity is mg pure test substance/kg bw/day. The recalculated NOAELs for maternal and developmental toxicity is 20.7 mg pure test substance/kg bw/day. The developmental effects are considered secondary to the maternal toxicity seen in the high dose group.