Registration Dossier

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2003-10-22 to 2004-02-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read-across from a guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 22, 2001
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Guidelines for Non-clinical Studies of Drugs Manual 1995; Guidelines for Toxicity Studies of Drugs. Japanese Ministry of Health and Welfare.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
certificate Stadt Hamburg, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

Test animals

Species:
rat
Strain:
other: CD/Crl:CD
Details on test animals and environmental conditions:
TEST ANIMALS
- Age (at day 0 of pregnancy): 8 - 9 weeks
- Weight (at day 0 of pregnancy): 205 - 254 g
- Fasting period before study: none
- Housing: singly in MAKROLON cages
- Diet: ad libitum, ssniff R-Z V1324, ssniff Spezialdiäten GmbH, D-59494 Soest, Germany
- Water : ad libitum, tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/-2 °C
- Humidity: relative 55% +/- 15%
- Photoperiod: 12 hours dark/12 hours light, 150 lux at app. 1.5 m room height

IN-LIFE DATES: From: Oct 22, 2003 To: Nov 19, 2003

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Control: 10 ml vehicle/kg bw /day
Dose levels: The dose levels referring to active ingredients were nominal 100, 300, and 1000 mg/kg bw/day and referring to test item 330, 990 and 3300 mg/kg bw/day. The nominal concentrations were analytically verified in samples taken at study initiation and study termination. The actual concentrations of the samples taken from the aqueous test item carrier mixtures were within the range of 101.9% to 109.9% of the nominal concentrations indicating correctly prepared application mixtures and a sufficient stability.

VEHICLE
- Justification for use and choice of vehicle (if other than water): aqua ad iniectabilaia
- Lot/batch no. (if required): 3175P13E/F, B. Braun Melsungen AG, D-34212 Melsungen, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the analysis of the concentration of the test item-carrier mixtures, samples of approx. 10 mL were taken at the following time-points and stored at -20°C or colder until analysis by LPT:
- At study initiation (1 st administration): Concentration and stability immediately after preparation of the mixture as well as 8 and 24 hours after storage of the test item preparations at room temperature (3 samples/dose level group). total number of samples: 9; Homogeneity at start of administration, during (middle) administration and before administration to the last animal of each dose level group (3 samples/dose level group). total number of samples: 9
- At termination of the administration period: Concentration during treatment with the test item always before administration to the last animal/dose level group (1 sample/dose level group). total number of samples: 3
The method used was made available by the sponsor and re-validated by LPT. Analytical Method for the Determination of C8-18 AAPB in Water with UV/VIS Detection. The following parameters were determined: linearity, accuracy, precision, sensitivity, specificity, stability.
The results of the analyses showed that the test item-carrier mixtures were correctly prepared and the concentration and stability found were in good agreement to those expected. The actual concentrations of the test item-carrier mixtures were within the measured range of 101.9% to 109.9% of the nominal C8-18 AAPB concentrations.

Details on mating procedure:
Sexually mature ('proved') male rats of the same breed served as partners. They were repeatedly employed, at the earliest two days after successful copulation. Females mated by the same male were placed in different groups (if possible). The female breeding partners were randomly chosen. Mating was monogamous: 1 male and 1 female animal were placed together in one cage during the dark period. Each morning a vaginal smear was taken to check for the presence of sperm and the stage of oestrus cycle. If findings were negative, mating was repeated. The day on which sperm was found was considered as the day of conception (day 0 of pregnancy). This procedure was repeated until enough pregnant dams were available for all groups. Rats which did not become pregnant were excluded from the analysis of the results and replaced by other animals. A post-mortem negative staining according to SALEWSKI was carried out in the replaced animals in order to confirm the non-pregnancy status.
Duration of treatment / exposure:
14 days, from 5th to the 19 th day of pregnancy, the day on which sperm was found was considered as the day of conception (day 0 of pregnancy).
Frequency of treatment:
daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300, 1000 mg/kg bw
Basis:
analytical conc.
based on a.i.
No. of animals per sex per dose:
Mated and treated animals: group 1 - 4: 25
Evaluated pregnant rats: group 1 - 4: 20 (the first 20 animals with pregnancy signs were used)
Animals evaluated for maternal toxicity: group: 1, 2, and 3: 20, group 4: 21 (one additional animal was included due to the premature death of one high-dosed dam)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected on available toxicity data of a former 14-day dose-range-finding to determine suitable dose
levels for a subsequent 90-day toxicity study.
- Rationale for animal assignment (if not random): the rat is a commonly used rodent species for embryotoxic studies
- Other: The test item was delivered as a 30% aqueous solution as it is marketed. To specify the impact of C8-18 AAPB the doses are calculated on the active ingredient. Whilst preparing the dosing solution a correction factor of 3.3 was used. Hence all dosing levels in this study refer to the active ingredient.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
-- Viability checks in addition to detailed clinical observation were made early in each working day and again in the afternoon to look for dead or moribund animals. This would have allowed post mortem examinations to be carried out during the working period of that day. On Saturdays and Sundays, a similar procedure was followed except that the final check was carried out at approximately midday.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
-- Individual animals were observed daily for behaviour, external appearance and nature of the faeces. Immediately after administration, any signs of illness or reaction to treatment were recorded. In case of changes, the animals were observed until the symptoms disappeared. In addition, animals were checked regularly throughout the working day from 7.00 a.m. to 3.45 p.m. On Saturdays and Sundays, the animals were checked regularly starting from 7.00 a.m. to 11.00 a.m. with a final check performed at approximately 3.30 p.m.

BODY WEIGHT: Yes
- Time schedule for examinations: recorded on day 0 of gestation, followed by daily weighings, always at the same time and carcass weight, once at termination
-- The weight of each rat was recorded on day 0 of gestation (the day of detection of a positive mating sign), followed by daily weighings - always at the same time of the day. The body weight gain was also calculated in intervals (i.e. 0-3, 3-6, 6-9, 9-12, 12-15,15-18 and 18-20). Furthermore the net weight change from day 6 was calculated (= carcass weight minus day 6 body weight; whereas carcass weight = terminal body weight minus uterine weight).
These measurements were also used for calculating the daily amount of test item to be administered.

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
-- The quantity of food consumed by each rat was recorded daily. Food intake per rat (g/rat/day) was calculated using the total amount of food given to and left by each rat in each group on completion of a treatment day. The relative food consumption (g/kg bw/day) was calculated using the following formula: Daily food consumption [g/kg bw/day] = Total food intake in g/Body weight in g * 1000

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily
-- Daily monitoring by visual appraisal of the drinking water bottles was maintained throughout the study.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: ovaries, uteri, internal organs, placentae
-- On the 20th day of gestation, the rats were laparotomised under ether narcosis. The ovaries and uteri were removed; the uteri (in toto) were weighed. In order to check for possible drug effects, a dissection with macroscopic examination of the internal organs and placentae of the dams was carried out on the day of scheduled laparotomy or on the day on which the animals were found dead. In case of macroscopical findings, the affected maternal tissues were preserved in 7% buffered formalin for possible future histopathological examinations. Macroscopic inspection (gross evaluation) of the placentae for example for focal indurations.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations:
- The number of fetuses (alive and dead) and placentae was determined.
- Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
- Number and size of resorptions were determined.
- Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
- The gravid uterus weight was determined.
- Weights of fetuses and weights of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the mean litter weight).
- Fetuses were inspected externally for damages, especially for malformations
The fetuses were sacrificed by an ether atmosphere.
Fetal examinations:
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes
-- 50% of the number of fetuses in each litter were examined for soft tissue anomalies. Body sections were made and examined according to WILSON.
- Skeletal examinations: Yes
-- 50% of the number of fetuses in each litter were examined for skeletal anomalies. The thorax and peritoneal cavity (without damage to ribs and sternum) were opened and the location, size and condition of the internal organs were determined. Then the skeleton was double-stained with Alcian blue for the examination of cartilage and with Alizarin red to reveal ossifications (according to DAWSON). The skeletal system was examined (determination of the number and type of retardations, variations as well as malformations).
- Head examinations: Yes
The fetuses were allocated to the evaluation of DAWSON or WILSON on an alternating basis.
Statistics:
Bartlett chi-square test for: numerical values, homogeneity of variances
Dunnet test: when variances were homogeneous, test was used to compare the experimental groups with control groups
Students test: in case of heterogeneity of variances
Fisher´s exact test: for comparison of classification measurements (i.g. malformation-, resorption-, retardation, and variation rate)

Indices:
- Corpora lutea: number per dam, absolute number per group, mean per group
- Implants: number per dam, distribution in the uterine horns, absolute number per group, mean per group
- Resorptions: number per dam, distribution in the uterine horns, absolute number per group, mean per group, mean % per group, early resorptions < 2mm, late resorptions > 2 mm
- Resorption rate [%] = (resorptions/implantations) x 100
- Weight of placentae: individual data per fetus, mean per litter, mean per group, litter mean per group, litter mean per sex and group
- Weight of fetuses: individual data per fetus, mean per litter, mean per sex and litter, litter mean per group, litter mean per sex and group
- Fetuses:number per dam (alive and dead), number of fetuses per sex and dam, distribution in the uterine horns, absolute number of fetuses alive per group, mean number of fetuses alive per group, mean % of fetuses alive per group, mean % per sex and group
- Dead fetuses: number per dam, mean per group
- Runts: number per dam, mean per group
- Malformed fetuses: individual data per fetus, mean per group and type of malformation
- Malformation rate per group [%] = (malformed fetuses/fetuses) x 100
- Fetuses with variations: individual data per fetus, mean per group and type of variation
- Variation rate per group [%] = (fetuses with variations/fetuses) x 100
- Fetuses with retardations: individual data per fetus, mean per group and type of retardation
- Retardation rate per group [%] = (fetuses with retardations/fetuses) x 100
- Pre-implantation loss [%] = ((corpora lutea - implantations)/corpora lutea) x 100
- Post-implantation loss [%] =((implantations - living fetuses)/implantations) x 100

Historical control data:
Summarized results of the 19 last embryotoxicity studies in Sprague-Dawley rats (Charles River Deutschland GmbH) performed at LPT in the years 2000 - 2003 were used as historical control data. These background data have not been audited by the Quality Assurance Unit of the testing facility.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced food consumption, impaired body weight and necropsy findings at 300 and 1000 mg a.i./kg bw/day
For further details see section "Any other information on results including tables "

Effect levels (maternal animals)

Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
100 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Reduced fetus weight and increased number of resorptions at 1000 mg a.i./kg bw/day
For further details see section "Remarks on results including tables and figures"

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
300 mg/kg bw/day
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Remarks:
based on a.i.
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

MATERNAL TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
Mortality: 100 and  300 mg a.i./kg bw / day: no mortality1000 mg a.i. /kg bw / day: 1/20 on gestation day 15

Clinical signs:

100 and 300 mg a.i./kg bw / day: no clinical signs of systemic toxicity

1000 mg a.i./kg bw /day: an abdominal position was noted in 13 of 21 dams on several (1 to 6) gestation days starting from the second administration day (gestation

day 6). This symptom started within 5 to 20 minutes after dosing and lasted for 20 minutes to 2 hours. Moreover, pilo-erection and reduced motility were noted in two

dams of the high dose group. This symptom was noted in one dam on gestation days 19 and 20 and in dam which died on gestation days 12 to 15 (until premature death).

The faeces of all dams were of normal consistency during the whole experiment..

Body weight:

100 and 300 mg a.i./kg bw / day: no test item-related influence on the body weight. A marginal, but statistically significant decrease by 4% to 5% was noted in the low

dose group (100 mg/kg bw / day) on gestation days 10 to 17 for the body weight. This is regarded to be spontaneous due to no comparable effects at 300 mg/kg

bw / day.

1000 mg a.i./kg bw / day: the body weight was moderately reduced (up to 17% below the control values, significant at p </= 0.01) from the start of treatment onwards until

laparotomy on gestation day 20.

Body weight change:

100 and 300 mg a.i./kg bw / day: No test item-related influence was noted on the body weight change.

1000 mg a.i./kg bw /day: the mean maternal body weight change was statistically significantly (at p </= 0.01) decreased on gestation days 3 to 6, 6 to 9, 12 to 15, 15

to 18 and 18 to 20.

Food and drinking water consumption:

100 mg a.i./kg bw / day: The food consumption was not influenced.

300 mg a.i./kg bw / day: The food consumption was marginally reduced on some days during the administration period (up to 12%, statistically significant on gestation days

8,10 and 19 at p </= 0.05 or p </=0.01).

1000 mg a.i./kg bw / day: A severely and statistically significantly (at </= 0.01 or p </= 0.05) reduced food consumption was noted from start of treatment until laparotomy,

predominantly during the first treatment days.

Drinking water consumption showed no test item-related changes in any of the treated groups as observed during daily visual appraisal.

EXAMINATION OF THE DAMS AT TERMINATION

Necropsy findings Stomach:

100 mg a.i./kg bw / day: No test item-related pathological findings.

300 mg a.i./kg bw / day: Thickened/partly thickened stomach mucosa in 4 of 20 animals, in addition ulcers (diameter approximately 1 mm or 0.5 to 1 mm) in 2/4 animals with thickened mucosa.

1000 mg a.i./kg bw /day: Thickened or partly thickened stomach mucosa (greyish discoloured in two dams) was noted in 20 of 21 dams including one prematurely deceased dam. In addition, in two of these dams a few ulcers were noted in the stomach (diameter up to 1 mm). These findings are regarded to be test item-related.

Necropsy findings other:

A reduced in size spleen was noted in one high-dosed dam (1000 mg/kg b.w./day) and is regarded to be an incidental finding.

Gravid Uterus weight:

100 and 300 mg a.i./kg bw / day: No test item-related influence.

1000 mg a.i./kg bw /day: A reduction by 22% (significant at p </= 0.01) was noted for the gravid uterus weight caused by the lowered fetal weights.

Carcass weight:

100 and 300 mg a.i./kg bw / day: No test item-related influence.

1000 mg a.i./kg bw / day: The carcass weight was statistically significantly (at p </= 0.01) reduced by 15% when compared with the control animals

Net body weight change from day 6 (= carcass weight minus day 6 body weight):

100 mg a.i./kg bw / day: No test item-related influence.

300 and 1000 mg a.i./kg bw / day:a statistically significant (at p </= 0.05 or p </= 0.01) reduction by 23% and 67%, respectively, was noted for the net weight change from day 6 onwards.


REPRODUCTION DATA OF DAMS (0, 100, 300, 1000 mg/kg bw):

No test item-related influence on the prenatal fetal development was detected at either 100, 300 or 1000 mg a.i. /kg bw / day with respect to the number of corpora lutea and implantation sites.

The number of resorptions (early, late and total) was increased in the high dose group (1000 mg/kg bw / day). Moreover, a statistically significant (p </= 0.01) increase was noted for the ratio of early, late and total resorptions to implantation sites in this dose group. As a result, the number of viable fetuses and the ratio of viable fetuses to implantation sites (statistically significant at p </= 0.01) were decreased at 1000 mg a.i. /kg bw / day. These changes are caused by a total post-implantation loss in two dams at an early time of pregnancy and are regarded to be test item related.

Detailed indices for test groups 0 (control), 100, 300 and 1000 mg a.i./kg bw / day, respectively:
- Corpora lutea: 301 (15.1 per dam), 313 (15.7 per dam), 311 (15.6 per dam), 316 (15.8 per dam)
- Implantation sites: 290 (14.5 per dam), 309 (15.5 per dam)*, 309 (15.5 per dam)**, 307 (15.4 per dam)
- Resorptions: 10 (0.5 per dam), 7 (0.4 per dam), 12 (0.5 per dam), 53 (2.7 per dam)**
- Early resorptions: 10 (0.5 per dam), 3 (0.2 per dam)*, 9 (0.5 per dam), 46 (2.3 per dam)**
- Late resorptions: 0 (0.0 per dam), 4 (0.2 per dam)*, 3 (0.2 per dam), 7 (0.4 per dam)**
- Live fetuses: 280 (14 per dam), 302 (15.1 per dam), 297 (14.9 per dam), 254 (14.1 per dam **, n= 18 dams with visible fetuses)

- Dead fetuses at laparotomy: in all test groups 0

- Pre-implantation loss (mean %): 6.3, 1.1, 0.6, 2.8

- Post-implantation loss (mean %): 3.3, 2.3, 4.2, 17.5

* Significantly different from control, p </= 0.05
** Significantly different from control, p </= 0.01

- comparing the ratio of implantation sites/corpora lutea of the test group with the ratio of implantation sites/corpora lutea of the control group

- comparing the ratio of resorptions/implantation sites of the test group with the ratio of resorptions/implantation sites of the control group

- comparing the ratio of fetuses/implantation sites of the test group with the ratio of fetuses/implantation sites of the control group

EXAMINATION OF THE FETUSES:

Sex distribution of fetuses:

100, 300 and 1000 mg a.i./kg bw / day: The sex distribution of the fetuses was comparable with that of the control fetuses.

Weight of placentae:

100, 300 and 1000 mg a.i./kg bw / day: The mean placental weights were not influenced by the administration of the test item to the dams when

compared with the control group.

Weight of fetuses:

100 and 300 mg a.i. /kg bw / day: The mean fetal weights were not influenced as compared to the control group.

1000 mg a.i./kg bw / day : The mean fetal weights calculated for male and female fetuses and for all fetuses were statistically significant (at p </= 0.01) below the control values. Although the mean fetal value of this group is still within the range of LPT background data, this effect is regarded to be test item-related.

External examination of the fetuses:

100, 300 and 1000 mg a.i./kg bw / day: No macroscopically visible malformations and variations were noted during external examination at laparotomy.

Laparotomy revealed no dead fetuses at any tested dose level. One runt each was noted at 100 and 300 mg/kg b.w./day. This change is regarded

to be spontaneous and is within the normal range of variation.

Skeletal examination of the fetuses:

100, 300 and 1000 mg a.i./ kg bw / day: The skeletal examination (according to DAWSON) revealed no malformed fetuses at any of the tested dose level and in the control group.

- The skeletal variations observed were related to the ribs (accessory 14th rib(s), rib(s) not ossified, shortened or wavy) and the sternum (sternebra(e) bipartite, dumbbell shaped or misaligned to a slight degree).

- No test item-related skeletal variations were noted at 100, 300 or 1000 mg a.i./kg bw / day.

- Although there was no statistical significance for the incidence of each variation in either dose group, a slight but statistically significant increase (at p </= 0.05) was seen in the total incidence of skeletal variations at 300 mg a.i./kg bw / day. This finding was judged as incidental as no dose-relationship was noted.

- Skeletal retardations were related to the 5th metacarpalia (not ossified), caudal vertebral bodies (only one body ossified), lumbar vertebral body/bodies (less than 6 ossified, bipartite), thoracic vertebral body/bodies (bipartite, dumbbell-shaped), hyoid (missing ossification), skull (incomplete ossification), sternebra(e) (incomplete or missing ossification, reduced in size).

- No test item-related influence was noted for the incidence of skeletal retardations at any of the tested dose levels (100, 300 or 1000 mg a.i./kg bw / day).

- Increased fetal and litter incidences (significant at p </= 0.05 or p </= 0.01) observed for not ossified 5th metacarpalia in all dosed groups are related to the low value obtained for the control group. Further, the observed incidences are still within the LPT background data of 0% to 7.8% mean fetal incidence for this retardation type.

- All other significances noted in the test item groups (fetal incidences for not or incompletely ossified hyoid, skull or sternebrae as well as fetal incidence for the total fetal skeletal retardations) are regarded to be without biological relevance, as these changes refer to a decrease in comparison with the control group.

Soft tissue examination of the fetuses:

100, 300 and 1000 mg a.i./kg bw / day: The examination of the fetal organs (according to WILSON) revealed no malformed fetuses at any of the tested dose level and in the control group.
- The fetal examination according to WILSON revealed the following variations: 4th cerebral ventricle dilated, cardiomegaly, dilated renal pelvis, misplaced kidney and
haemorrhage / haemorrhagic focus/foci in the liver or thoracic cavity. No statistically significant differences in fetal or litter incidences were noted for these variations at any of the tested dose levels (100, 300 or 1000 mg/kg b.w./day). These findings are very common in the rat strain used and the incidences observed were within the historical background range.

Detailed indices for test groups 0 (control), 100, 300 and 1000 mg a.i./kg bw / day, respectively:

- Malformations (external, skeletal, soft tissue) (fetal incidence): in all test groups 0
- External variations (fetal incidence): in all test groups 0
- Skeletal variations (fetal incidence):5, 8, 13*, 6 (finding was judged as incidental as no dose-relationship was noted)
- Skeletal retardations (fetal incidence): 129, 137, 130, 125
- Soft Tissue variations (fetal incidence): 8, 12, 10, 9

* Significantly different from control, p </= 0.05


Applicant's summary and conclusion

Conclusions:
In this developmental toxicity / teratogenicity study, performed according to OECD TG 414 on CD rats, 330, 990 and 3300 mg/kg bw/day of a 28.9 % aqueous C8-18 AAPB solution, corresponding to 100, 300, and 1000 mg active substance/kg bw/day, respectively, were applied by gavage. Dose-related maternal toxic effects (reduced food consumption, impaired body weight and necropsy stomach findings) occurred at 990 mg/kg bw/day and above. Embryotoxic effects (reduced mean fetal weight and increased number of resorptions) were found only at the maternal toxic dose level of 3300 mg/kg bw/day. Up to and including the highest tested dose, no external, skeletal or soft tissue malformations and no external variations were found. The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active substance/kg bw/day) and the NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active substance/kg bw). The NOEL for teratogenic effects was the highest tested dose of 3300 mg/kg bw/day, corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.
Executive summary:

In a developmental toxicity study according OECD 414, C8 -18 AAPB (28.9 % a.i, 62 % water, and 5.4 % NaCl) was administered to 25 females CD rats/dose at dose levels of 0, 330, 990, 3300 mg from day 5 through 19 of gestation by gavage. The test item dose levels refer to nominal active ingredient of 100, 300, and 1000 mg/kg bw/day. The nominal values were analytically verified in samples taken at study initiation and study termination. The actual concentrations of the samples taken from the aqueous test item carrier mixtures were within the range of 101.9% to 109.9% of the nominal C8 -18 AAPB concentrations indicating correctly prepared application mixtures and a sufficient stability. Number of evaluated pregnant rats were 20/group (the first 20 animals with pregnancy signs were used). Animals evaluated for maternal toxicity were 20/group except of high dose group in which one additional animal was included due to a premature death of one dam.

Regarding maternal toxicity, the dams of the 990 mg/kg bw/day group showed decreased net body weight change from day 6 onward (= carcass weight minus day 6 body weight), reduced food consumption, thickened/partly thickened stomach mucosa in 4 of 20 animals and in addition ulcers (diameter approximately 1 mm or 0.5 to 1 mm) in 2/4 animals with thickened mucosa. In the 3300 mg/kg bw/day group the dams showed severely reduced food consumption, reduced body weights (absolute, body weight gain on gestation days 3 to 6, 6 to 9, 12 to 15, 15 to 18 and 18 to 20, and net body weight change from day 6 onward), reduced carcass weight and reduced gravid uterus weights. Thickened or partly thickened stomach mucosa (greyish discoloured in two dams) was noted in 20 of 21 dams including one prematurely deceased dam. In addition, in two of these dams a few ulcers were noted in the stomach (diameter up to 1 mm).

The number of early, late and total resorptions was increased in the 3300 mg/kg bw/day group, and the ratio of viable fetuses to implantation sites was decreased compared to the controls. This was due to a total post-implantation loss in two dams in this dose group. In addition, a statistically significant reduction in fetal weights and in the number of viable fetuses as compared to the control was observed. No external, skeletal or soft tissue malformations and no external variations were found. The NOEL for maternal toxicity was 330 mg/kg bw/day (corresponding to 100 mg active ingredient/kg bw/day). The NOEL for developmental toxicity was 990 mg/kg bw/day (corresponding to 300 mg active ingredient/kg bw/day).

The NOEL for external, skeletal or soft tissue malformations and variations was the highest tested dose of 3300 mg/kg bw/day (corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.

The developmental toxicity study in the rat is classified acceptable and satisfies the guideline requirement for a developmental toxicity study in rat.