C12-14 (even numbered) alkyl glycosides, oligomeric and C12-14 (even numbered) alkyl glycosides, oligomeric, carboxymethyl ethers, sodium salts

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C12-14 (even numbered) alkyl glycosides, oligomeric and C12-14 (even numbered) alkyl glycosides, oligomeric, carboxymethyl ethers, sodium salts

EC Number:

701-129-1

Molecular formula:

C18.4 H27.8 O8.4; C20.4, H31.8 O8.4; C22.4 H35.8 O8.4; C24.4 H39.8, O 8.4; C20.4 H29.8 O10.4; C22.4 H33.8 O10.4; C24.4 H37.8 O10.4; C26.4 H41.8 O10.4; C22.4 H31.8 O 12.4, C24.4 H35.8 O12.4; C26.4 H39.8 O12.4; C28.4 H43.8 O12.4; C24.4 H33.8 O14.4; C26.4 H37.8 O14.4; C28.4 H41.8 O14.4; C30.4 H45.8 O14.4, all formulas are generic according oligomeric structure!

IUPAC Name:

C12-14 (even numbered) alkyl glycosides, oligomeric and C12-14 (even numbered) alkyl glycosides, oligomeric, carboxymethyl ethers, sodium salts

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0018286787
- Expiration date of the Batch: 11 Dec 2018
- Content: 33.8 g/100 g (100 g/100 g minus water content)
- Physical state / appearance: Liquid / yellowish to beige, turbid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator
- Solubility and stability of the test substance in the solvent/vehicle: Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was dissolved.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI [Han]

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10 - 12 weeks
- Weight at study initiation: The body weight of the pregnant animals on day 0 varied between 147.4 - 200.1 g.
- Housing: individually in Polycarbonate cages type III
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse / rat "GLP", meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): potable tap water; ad libitum
- Acclimation period: between start of the study (beginning of the experimental phase) and first administration (GD 6)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 45 - 65 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature.
For the test substance preparations, the specific amount of test substance was weighed, topped up with deionized water in a graduated flask and intensely mixed with a magnetic stirrer until it was dissolved.
Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in deionized water over a period of 7 days at room temperature had been verified prior to the start of the study in a similar batch.
Samples of the test substance preparations were sent (at the beginning of administration and due to equivocal analytical results) to the analytical laboratory for verification of the concentrations. The samples taken for the concentrations control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (300 and 3000 mg/kg bw/day). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.
All test samples, plus a duplicate set of reserve samples, were withdrawn by staff of the Reproduction Toxicology. All reserve samples and further samples were stored at the Laboratory Reproduction Toxicology frozen (at - 20 °C). Analysis of these samples were performed in case of equivocal analytical results with the original samples or after loss of / damage to original samples after agreement by the Study Director.

Details on mating procedure:

- Impregnation procedure: The animals were paired by the breeder ("time-mated")
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

from implantation to one day prior to the expected day of parturition (GD 6 to GD 19)

Frequency of treatment:

once daily

Duration of test:

14 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

corresponds to 100 mg/kg bw/day active ingredient

Doses / concentrations 2

Dose / conc.:

900 mg/kg bw/day (nominal)

Remarks:

corresponds to 300 mg/kg bw/day active ingredient

Doses / concentrations 3

Dose / conc.:

3 000 mg/kg bw/day (nominal)

Remarks:

corresponds to 1000 mg/kg bw/day active ingredient

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen at the request of the sponsor. The oral route was selected since this has proven to be suitable for the detection of a toxicological hazard.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily before and after treatment period (GD 0 - 5 and 20); before administration as well as within 2 hours and within 5 hours after administration during treatment period (GD 6 - 19)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: GD 0, 1, 3, 6, 8, 10, 13, 15, 17, 19 and 20

FOOD CONSUMPTION: Yes
- Time schedule for examinations: The consumption of food was recorded for the intervals GD 0 - 1, 1 - 3, 3 - 6, 6 - 8, 8 - 10, 10 - 13, 13 - 15, 15 - 17, 17 - 19 and 19 - 20

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: uteri and ovaries

OTHER: mortality

Ovaries and uterine content:

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

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
- Skeletal examinations: Yes
- Head examinations: No

In the present study the internationally harmonized glossary of WISE et al. (1997) and the updated version of MAKRIS et al. (2009) was essentially used to describe findings in fetal morphology. Classification of these findings was based on the terms and definitions proposed by CHAHOUD and SOLECKI:

Malformation
A permanent structural change that is likely to adversely affect the survival or health.

Variation
A change that also accurs in the fetuses of control animals and / or is unlikely to adversely affect the survival or health. This includes delays in growth or morphogenesis that have otherwise followed a normal pattern of development.

The term "unclassified observation" was used for those fetal findings, which could not be classified as malformations or variations.

Statistics:

DUNNETT-test (two-sided): Food consumption, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, propportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight

FISHER'S EXACT test (one-sided): Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings

WILCOXON-test (one-sided): Proportions of fetuses with malformations, variations and / or unclassified observations in each litter

Indices:

sex ratio
conception rate (in %)
preimplantation loss (in %)
postimplantation loss (in %)

Historical control data:

Included in the final report.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Nearly all (23 out of 25) females of the high-dose group (3000 mg/kg bw/day) and four (out of 25) females of the mid-dose group (900 mg/kg bw/day) showed occasional salivation during the treatment period. Salivation occurred in the respective animals only shortly, i.e. within 0 - 2 h, after treatment and was observed during GD 7 - 19 (test group 3) or GD 17 - 19 (test group 2). No clinical signs or changes of general behavior were detected in any female of all test groups beyond 2 hours after treatment. The occasional salivation was most probably caused by the bad taste or smell of the test substance and was not assessed as sign of systemic toxicity.
No further clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any female at dose levels of 300, 900 or 3000 mg/kg bw/day during the entire study period.

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Description (incidence):

There were no test substance-related or spontaneous mortalities in any females of all test groups (0, 300, 900 or 3000 mg/kg bw/day).

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

The mean body weights of the high-dose dams (3000 mg/kg bw/day) were comparable to the concurrent control group throughout the entire study period.
The mean body weight change in this group was statistically significantly reduced during one single measurement period (GD 10 - 13). As it was a temporary, single deviation it was assessed as incidental and not toxicological relevant.
The mean body weights and the average body weight gain of all dams (300 and 900 mg/kg bw/day) were generally comparable to the concurrent control group throughout the entire study period.
The corrected body weight gain of test groups 1, 2 and 3 (300, 900 and 3000 mg/kg bw/day) revealed no difference of any biological relevance to the corresponding control group. Moreover, mean carcass weights of all test groups remained unaffected by the treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

The mean food consumption of the high-dose dams (3000 mg/kg bw/day) was comparable to the concurrent control group if calculated for the entire treatment period (GD 6 - 19). In this dose group the mean food consumption was statistically significantly reduced during one single measurement period (GD 10 - 13 about 10 % below control). As it was a slight, single deviation it was assessed as incidental and not toxicological relevant.
The mean food consumption of the mid- and low-dose dams (900 and 300 mg/kg bw/day) was generally comparable to the concurrent control group throughout the entire study period.

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

Description (incidence and severity):

The mean gravid uterus weights of the animals of test groups 1 - 3 (300, 900 and 3000 mg/kg bw/day) were not influenced by the test substance. The differences between these groups and the control group revealed no dose-dependency and were assessed to be without biological relevance.
The mean placental weights of test groups 1 - 3 were comparable to the concurrent control group.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No necropsy findings which could be attributed to the test substance were seen in any dam.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Maternal developmental toxicity

Number of abortions:

not examined

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There were no test substance-related and / or biologically relevant differences between the different test groups in the values calculated for the pre- and post-implantation losses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

There were no test substance-related and / or biologically relevant differences between the different test groups in the number of resorptions. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Description (incidence and severity):

There were no test substance-related and / or biologically relevant differences between the different test groups in the number of viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
One dead fetus was found at cesarean section of mid-dose dam No. 74 (900 mg/kg bw/day) which is a rare finding but may occur spontaneously in this rat strain.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The conception rate was 96 % in the mid-dose group (900 mg/kg bw/day) and 100 % in the control, the low- and high-dose groups (0, 300 and 3000 mg/kg bw/day).

Other effects:

no effects observed

Description (incidence and severity):

There were no test substance-related and / or biologically relevant differences between the different test groups in the mean number of corpora lutea and implantation sites. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.

Effect levels (maternal animals)

1

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Basis for effect level:

other: no evidence of maternal toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Basis for effect level:

other: no evidence of maternal toxicity

Maternal abnormalities

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There were no test substance-related and / or biologically relevant differences between the different test groups in the number of viable fetuses. All observed differences are considered to reflect the normal range of fluctuations for animals of this strain and age.
One dead fetus was found at cesarean section of mid-dose dam No. 74 (900 mg/kg bw/day) which is a rare finding but may occur spontaneously in this rat strain.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1 - 3 (300, 900 and 3000 mg/kg bw/day) was comparable to the control fetuses.

Changes in litter size and weights:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

External malformations were detected just in one dead fetus in test group 2 (900 mg/kg bw/day). In this case, the external malformations, i.e. meningocele, an- and microphthalmia, cleft plate, curled tail, were associated with skeletal malformations. None of these malformations were assessed as treatment-related since they were not related to dose and the finding "multiple external malformations" can be found in the historical control data at comparable incidences.
The total incidence of external malformations in treated animals did not differ significantly from the concurrent control group and was covered by the historical control data.
No external variations were recorded.
No external unclassified observations were recorded.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were noted in two fetuses, each, in test groups 1, 2 and 3 (300, 900 and 3000 mg/kg bw/day). One dead fetus of the mid-dose group (No. 74-08) had multiple skeletal malformations concerning the skull, the vertebral column and ribs. The malformations were not assessed as treatment-related since they occurred in single fetuses without a relation to dose.
The total incidences of skeletal malformations in treated animals did not differ significantly from the control group and were comparable to the historical control data.
For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and appeared without a relation to dose. The overall incidences of skeletal variations were comparable to the historical control data. The increased incidences of skeletal variations were either not related to the dose or they were inside the historical control range. Therefore, they are not considered as treatment-related.
Additionally, some isolated cartilage findings without impact on the respective bony structures, which were designated as inclassified cartilage observations, occurred in all test groups. The observed unclassified cartilage findings were related to the skull, the vertebral column, the ribs and the sternum and did not show any relation to dosing. However, the incidence of "notched manubrium" was significantly increased in test group 1 (300 mg/kg bw/day). As this finding both showed no dose-dependency and could be found in the historical control data, it was assessed to be without biological relevance.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

No soft tissue malformations were recorded.
Three soft tissue variations were detected, i.e. misaligned palatal rugae in test group 2, dilated renal pelvis in all test groups and dilated ureter in test groups 0 and 2. The incidences of these variations were neither statistically significantly nor dose-dependently increased in the treated groups. All of them can be found in the historical control data at comparable incidences. Therefore, they were not assessed as treatment-related.
No soft tissue unclassified observations were recorded.

Effect levels (fetuses)

1

Dose descriptor:

NOAEL

Effect level:

3 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no evidence of developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no evidence of developmental toxicity

Fetal abnormalities

Abnormalities:

effects observed, non-treatment-related

Overall developmental toxicity

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Individual fetal external malformations

Test group	Dam No. – Fetus No., Sex	Finding
0 (0 mg/kg bw/day)	None
1 (300 mg/kg bw/day)	None
2 (900 mg/kg bw/day)	74-08 D^a)	Multiple external malformations
3 (3000 mg/kg bw/day)	None

D dead

^a) fetus with additional skeletal malformation

Table 2: Total external malformations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 253	25 268	24 258	25 260
Fetal incidence	N (%)	0.0	0.0	1 (0.4)	0.0
Litter incidence	N (%)	0.0	0.0	1 (4.2)	0.0
Affected fetuses/litter	Mean %	0.0	0.0	0.3	0.0

N number

Table 3: Total soft tissue variations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 119	25 130	24 123	25 123
Fetal incidence	N (%)	3 (2.5)	4 (3.1)	3 (2.4)	3 (2.4)
Litter incidence	N (%)	3 (12)	4 (16)	3 (13)	3 (12)
Affected fetuses/litter	Mean %	6.0	2.8	2.8	2.8

N number

Table 4: Individual fetal skeletal malformations

Test group	Dam No. – Fetus No., Sex	Finding
0 (0 mg/kg bw/day)	None
1 (300 mg/kg bw/day)	29-07 M 41-01 F	Fused rib Additional vertebral arch and corresponding rib
2 (900 mg/kg bw/day)	74-08 D^a) 75-01 M	Multiple skeletal malformations Shortened scapula
3 (3000 mg/kg bw/day)	97-01 M 97-05 M	Shortened humerus Small cervical arch

M male

F female

D dead

^a) fetus with additional external malformations

Table 5: Total skeletal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 134	25 138	24 135	25 137
Fetal incidence	N (%)	0.0	2 (1.4)	2 (1.5)	2 (1.5)
Litter incidence	N (%)	0.0	2 (8.0)	2 (8.3)	1 (4.0)
Affected fetuses/litter	Mean %	0.0	1.2	1.3	2.0

N number

Table 6: Total fetal skeletal variations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 134	25 138	24 135	25 137
Fetal incidence	N (%)	130 (97)	132 (96)	131 (97)	132 (96)
Litter incidence	N (%)	25 (100)	25 (100)	24 (100)	25 (100)
Affected fetuses/litter	Mean %	96.9	96.0	96.7	96.3

N number

Table 7: Occurrence of statistically significantly increased fetal skeletal variations (expressed as mean percentage of affected fetuses/litter)

Finding	Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d	HCD Mean % (range)
Supernumerary thoracic vertebra	3.5	12.3*	1.7	4.1	4.1 (0.8 – 11.0)
Supernumerary rib (14^th); cartilage present	5.0	14.5*	6.4	2.5	7.3 (1.9 – 14.7)

HCD Historical control data

* p ≤ 0.05 (Wilcoxon-test [onesided])

** p ≤ 0.01 (Wilcoxon-test [one-sided])

Table 8: Total unclassified cartilage observations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 134	25 138	24 135	25 137
Fetal incidence	N (%)	103 (77)	104 (75)	105 (78)	102 (74)
Litter incidence	N (%)	25 (100)	25 (100)	24 (100)	24 (96)
Affected fetuses/litter	Mean %	76.9	76.0	77.2	75.4

N number

Table 9: Total fetal malformations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 253	25 268	24 258	25 260
Fetal incidence	N (%)	0.0	2 (0.7)	2 (0.8)	2 (0.8)
Litter incidence	N (%)	0.0	2 (8.0)	2 (8.3)	1 (4.0)
Affected fetuses/litter	Mean %	0.0	0.6	0.7	1.1

N number

Table 10: Total fetal variations

		Test group 0 0 mg/kg bw/d	Test group 1 300 mg/kg bw/d	Test group 2 900 mg/kg bw/d	Test group 3 3000 mg/kg bw/d
Litter Fetuses	N N	25 253	25 268	24 258	25 260
Fetal incidence	N (%)	133 (53)	136 (51)	134 (52)	135 (52)
Litter incidence	N (%)	25 (100)	25 (100)	24 (100)	25 (100)
Affected fetuses/litter	Mean %	54.3	50.9	52.1	52.1

N number

Applicant's summary and conclusion

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6 - 19) at doses as high as 3000 mg/kg bw/day (corresponds to the limit dose of 1000 mg/kg bw/day active ingredient) caused neither evidence of maternal nor developmental toxicity.
In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 3000 mg/kg bw/day (1000 mg/kg bw/day active ingredient).

Executive summary:

In a prenatal developmental toxicity study, the test substance was tested for its prenatal developmental toxicity in Wistar rats. The test substance was administered as an aqueous preparation to groups of 25 time-mated female Wistar rats by gavage at doses of 300, 900 and 3000 mg/kg bw/day on gestation days (GD) 6 through 19. The administered doses corresponded to 100, 300 and 1000 mg/kg bw/day active ingredient (based on a 33.8 % active ingredient content of the test item). The control group, consisting of 25 females, was dosed with the vehicle (deionized water) in parallel. A standard dose volume of 10 mL/kg bw was used for each test group.

Analyses confirmed the correctness of the prepared concentrations, the homogeneous distribution and the stability of the test substance in the vehicle.

At terminal sacrifice on GD 20, 24 - 25 females per group had implantation sites.

Food consumption and body weights of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day.

On GD 20, all surviving females were sacrificed by decapitation (under isoflurane anesthesia) and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each dam, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for external findings. Thereafter, one half of the fetuses of each litter were examined for soft tissue findings and the remaining fetuses for skeletal (inclusive cartilage) findings.

The stability of the test substance preparations over a period of 7 days at room temperature was demonstrated.

The homogeneous distribution of the test substance in the vehicle was shown.

The correctness of the prepared concentrations was shown.

Generally, clinical observations including food consumption and body weight gain revealed no toxicologically relevant difference between the animals receiving 300, 900 or 3000 mg/kg bw/day of the test substance and controls.

Nearly all (23 out of 25) females of the high-dose group (3000 mg/kg bw/day) and four (out of 25) of the mid-dose group (900 mg/kg bw/day) showed transient salivation during the treatment period. Salivation persisted in the respective animals only for some time after daily gavage dosing (maximum up to 2 hours) and was initially observed on GD 7 (test group 3) and GD 17 (test group 2). Transient salivation shortly after dosing most likely reflects a reaction of the animals to the taste and smell of the test substance. It is not considered to be a sign of systemic toxicity and was, therefore, not assessed as treatment-related and adverse.

No differences of toxicological relevance between the control and the treated groups (300, 900 or 3000 mg/kg bw/day) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. Similarly, no influence of the test substance on fetal weight and sex distribution of the fetuses was noted at any dose.

Overall, there was no evidence for toxicologically relevant adverse effects of the test substance on fetal morphology at any dose.

The following test substance-related adverse effects / findings were noted:

- Test group 3 (3000 mg/kg bw/day - corresponds to 1000 mg/kg bw/day active ingredient): No test substance-related adverse effects on dams, gestational parameters or fetuses.

- Test group 2 (900 mg/kg bw/day - corresponds to 300 mg/kg bw/day active ingredient): No test substance-related adverse effects on dams, gestational parameters or fetuses.

- Test group 1 (300 mg/kg bw/day - corresponds to 100 mg/kg bw/day active ingredient): No test substance-related adverse effects on dams, gestational parameters or fetuses.

Under the conditions of this prenatal developmental toxicity study, the oral administration od the test substance to pregnant Wistar rats from implantation to one day prior to the expected day of parturition (GD 6 - 19) at doses as high as 3000 mg/kg bw/day (corresponds to the limit dose of 1000 mg/kg bw/day active ingredient) caused neither evidence of maternal nor developmental toxicity.

In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is the highest tested dose of 3000 mg/kg bw/day (1000 mg/kg bw/day active ingredient).