Registration Dossier

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Link to relevant study records

Referenceopen allclose all

Endpoint:
fertility, other
Remarks:
assessment of reproduction organs during Repeated Dose 90-Day Oral Toxicity in Rodents
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see "General Justification for Read-Across" attached to IUCLID section 13

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Mutual read across from the AAPBs to one another is justified:

a) Based on the information given in section 1, it can be concluded that all AAPBs mentioned above are similar in structure, since they are manufactured from similar resp. identical precursors under similar conditions and all contain the same functional groups. Thus a common mode of action can be assumed.
b) The content of minor constituents in all products are comparable and differ to an irrelevant amount.
c) The only deviation within this group of substances is a minor variety in their fatty acid moiety, which is not expected to have a relevant impact on intrinsic toxic or ecotoxic activity and environmental fate. Potential minor impact on specific endpoints will be discussed in the specific endpoint sections.

The read-across hypothesis is based on structural similarity of target and source substances. Based on the available experimental data, including key physico-chemical properties and data from toxicokinetic, acute toxicity, irritation, sensitisation, genotoxicity and repeated dose toxicity studies, the read-across strategy is supported by a quite similar toxicological profile of all five substances.
The respective data are summarised in the data matrix; robust study summaries are included in the Technical Dossier in the respective sections.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
see "General Justification for Read-Across" attached to IUCLID section 13

3. ANALOGUE APPROACH JUSTIFICATION
see "General Justification for Read-Across" attached to IUCLID section 13

4. DATA MATRIX
see "General Justification for Read-Across" attached to IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: There were no adverse test substance-related effects on reproductive organs (organ weights of ovary and testis and histopathology of gonads) up to the highest tested dose level.
Key result
Critical effects observed:
no
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Remarks on result:
not measured/tested
Key result
Reproductive effects observed:
no
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
An Extended one-generation reproductive toxicity study (Annex X, Section 8.7.3.; test method: EU B.56./OECD TG 443) in rats, oral route was requested with the substance (carboxymethyl)dimethyl-3-[(1-oxododecyl)amino]propylammonium hydroxide (C12 AAPB, CAS no 4292-10-8, EC no 224-292-6).

Since no systemic toxicity studies were available for the test substance so far, preliminary studies were initiated, including a Reproduction/Developmental toxicity screening test in rats (OECD TG 421), which was supposed to provide the necessary information to select appropriate dose levels for the subsequent Extended One-Generation Reproductive Toxicity Study (OECD TG 443).

However, due to unexpected mortality and adverse effects at the very beginning of the treatment period (mortality of 8/10 males and 3/10 females in the high dose group and severe local effects in the stomach in mid dose group in 6/10 males, 10/10 females), it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.
Therefore, in agreement with the Sponsor, it was decided to interrupt the treatment at 1000 mg/kg/day from Day 3 of the study and to investigate only the toxicity of the test item, C12 AAPB, changing the study design of the OECD TG 421 study to a 2-week oral preliminary toxicity study.
Additional studies were initiated to further investigate the reasons for this unexpectedly high local toxicity and to determine suitable dose levels for the OECD TG 421 and subsequent OECD TG 443 study.

Further details on the delay are attached below.

Reproductive effects observed:
not specified
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD guideline studies, RL1, GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A preliminary toxicity study was conducted on the source substance C12 AAPB as requested by ECHA: Initially, the aim of this study was to obtain information on the possible toxic effects on Sprague Dawley rats of both sexes after repeated dosing with C12 AAPB, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring in agreement with the OECD guideline 421. The animals, 10 per sex, were assigned to four groups and administered orally, by gavage, at dose levels of 100, 300 and 1000mg/kg/day. Control animals received the vehicle (softened water by reverse osmosis) at the same dose volume of 10mL/kg.

Due to unexpected mortality and adverse effects at the very beginning of the treatment period, it seemed evident that pairing and subsequent gestation and parturition of animals could be affected by the observed toxicity.

Therefore, in agreement with the Sponsor, it was decided to interrupt the treatment at 1000 mg/kg/day from Day 3 of the study and to investigate only the toxicity of the test item, C12 AAPB, changing the current study design to a 2-week oral preliminary non GLP study.

 

Mortality

Eleven cases of premature death, 8 males and 3 females receiving 1000mg/kg/day (Group 4) were found dead on Day 2 of treatment or, in one instance (one male), sacrificed for humane reasons on Day 3 of treatment.

Ataxia, decreased activity, hunched posture (kyphosis), staining of the ventral region, piloerection and salivation were the treatment-related clinical signs recorded in all these animals just prior their deaths. At the post mortem examination, yellow, brown and/or red staining of ventral or urogenital region and/or muzzle and dark and/or red colour of brain were observed. All the reported macroscopic observations were considered contributory to the cause of deaths.

 

Clinical signs

Piloerection and salivation were the two most relevant treatment-related clinical signs recorded in male and female animals dosed at 300mg/kg/day. The signs were considered treatment related but not adverse.

 

Body weight and body weight gain

Changes noted in body weight gain in groups 2 and 3 were considered incidental or not adverse.

 

Food consumption

No differences in food consumption were recorded during the study in animals of Groups 2 and 3, when compared to controls.

 

Terminal body weight and organ weights

No changes were observed in terminal body weight, or absolute and relative organ weights of control and treated animals that completed the treatment or in animals of Group 4 after 14 days of the recovery period.

 

Macroscopic observations

The most relevant change observed in rats treated at 300 and 1000mg/kg/day of both sexes was thickened stomach (non glandular region), when compared to controls.

 

Based on the results obtained in this study, it can be concluded:

– The dose level of 1000 mg/kg/day induced an acute toxicity when administered to Sprague Dawley rats as demonstrated by mortality and severe signs such ataxia, hunched posture and decreased activity. Treatment related findings described as thickened stomach (non glandular region), were observed in animals after 12 days of recovery period.

– The dose level of 300 mg/kg/day induced treatment related clinical signs such as piloerection and salivation, reduction in body weight gain and thickened stomach (non glandular region) observed at the macroscopic examination. These signs were considered treatment-related, but not adverse.

 

The dose level of 300 mg/kg/day is considered the NOAEL (No Observed Adverse Effect Level) for this study.

 

 

Conclusion

When the study data are available, a robust study summary will be prepared and submitted within an update of the dossier. Evaluation will be reconsidered based on the outcome of the extended one generation reproduction toxicity study.

 

 

Effects on developmental toxicity

Description of key information
A relevant, reliable and adequate developmental toxicity / teratogenicity study on C8-18 AAPB is available. In this study, performed according to OECD TG 414 on CD rats, 330, 990 and 3300 mg/kg bw/day of a 28.9 % aqueous solution of C8-18 AAPB, 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.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-10-22 to 2004-02-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
Jan 22, 2001
Deviations:
no
Qualifier:
according to guideline
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. QA statement)
Remarks:
certificate Stadt Hamburg, Germany
Limit test:
no
Species:
rat
Strain:
other: CD/Crl:CD
Details on test animals or test system 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
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
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
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.
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.
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 "
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
no effects observed
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"
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day
Based on:
act. ingr.
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
no effects observed
Key result
Developmental effects observed:
no
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


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.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guideline study, RL1, GLP
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a prenatal developmental toxicity study performed 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/kb bw/day (corresponding to the guideline limit dose of 1000 mg active ingredient/kg bw/day.

 

In HERA risk assessment report first edition, 2005 an additional developmental toxicity study is reported as follows: “One further developmental toxicity study is available with cocamidopropyl betaine (30% active substance). Female pregnant rats were administered 0, 30, 90 or 300 mg/kg bw on days 6 through 17 of gestation. No treatment-related effects on the incidence of fetal external, visceral, or skeletal malformations or developmental variations were observed among litters from dams in any of the treated groups.

The maternal and developmental no-observed-effect level (NOEL) of this study was 300 mg/kg bw/d, the highest level (Colgate-Palmolive, 2000).”

 

 

A 2 week preliminary toxicity study with C12 AAPB was conducted in New Zealand White (NZW) female rabbits with particular aim to investigate its potential local gastric irritating effect.

Female rabbits (4/dose) received the test item, dissolved in softened water, at dose levels of 100, 300 and 600 mg/kg/day (in terms of test item corrected for purity (30.43%)) by oral gavage. The dose volume was set at 5mL/kg. Control animal received softened water as vehicle during the same treatment period. The animals were checked twice a day for mortality and daily monitored for clinical signs. The bodyweight and food consumption were measured twice a week. Detailed macroscopic examination, organ weights and histopathology of the gastrointestinal tract, were carried out.

 

Mortality

On Day 3, two females at 600mg/kg/day, were found dead and the remaining females were sacrificed for humane reasons. On Day 5, one female at 300mg/kg/day was found dead and the remaining females were sacrificed for humane reasons.

At necropsy, the findings affecting the stomach could be considered the factor contributory to the ill status or death of these animals.

No mortality occurred at 100mg/kg/day.

 

Clinical signs

Severe clinical signs including dyspnoea, pale appearance, red nasal discharge, coldness to touch, brown staining in the perianal region and decreased activity were recorded at 300 and 600mg/kg/day. Reduced or liquid faeces were observed in the cage.

Reduced faeces and reduced food intake were recorded at 100mg/kg/day. Dyspnoea, transparent nasal discharge, rales and emaciated appearance were also observed in one low dose female.

 

Body weight

On Day 5, marked reduced body weight was observed in females dosed at 300mg/kg/day (-15% respect to Day 1). Body weight was reduced at 100mg/kg/day, especially in two females.

 

Food consumption

Severe reduced food consumption was noted at 300mg/kg on Day 5. A decrease in food consumption was recorded at 100mg/kg/day starting from Day 5 onward.

 

Terminal body weight and organ weights

A slight decrease was observed in terminal body weight of low dose females sacrificed at term when compared to controls. The statistically significant decrease in absolute and relative uterus weight was considered of doubtful interpretation, as not corroborated by histopathological evaluation.

 

Macroscopic observations

Treatment-related changes were noted in the pyloric region of the stomach of a single low dose female rabbit and was characterised by the presence of multiple pinpoint red areas associated with a single depressed dark red area. The macroscopic changes correlated at histopathological examination with erosion, haemorrhage and chronic inflammation.

 

Microscopic observations

At final sacrifice, treatment-related findings were seen in the stomach of one low dose female and consisted of erosion of the mucosal epithelium associated with haemorrhage and chronic inflammation of tunica muscularis and/or submucosal of fundic region of the stomach.

 

Conclusion

In conclusion, based on the results obtained in this investigative study, signs of toxicity were seen at all dose levels. The treatment-related findings at histopathological examination of the stomach confirmed the potential gastric irritant effect of C12 AAPB.

 

Based on the results of this study, the potential maximum dose level of C12 AAPB for a prenatal developmental toxicity study in rabbit would be 100 mg/kg bw/d or even lower. This is no meaningful highest dose level for an otherwise relatively non-toxic substance.

 

In addition, Braakhuis et al. (2019) found, that potential interspecies differences in developmental N(L)OAEL are in the same range as the interstudy reproducibility error, making the added value of a study conducted in a second species questionable. The authors have analysed a database with rat and rabbit developmental toxicity studies for over 1000 industrial chemicals, pesticides, veterinary drugs and human pharmaceuticals, which included 143 compounds with multiple oral rat studies and 124 compounds with multiple oral rabbit studies. This analysis demonstrated, that on average over all compounds, rat and rabbit do not differ in sensitivity towards developmental effects.

 

Reference:

Braakhuis HM et al. (2019) Testing developmental toxicity in a second species: are the differences due to species or replication error? Regulatory Toxicology and Pharmacology 107 (2019) 104410

 

There are no data gaps for the endpoint developmental toxicity. No human data are available. However, there is no reason to believe that these results from rabbit would not be applicable to humans.

Justification for classification or non-classification

There is no evidence for an intrinsic toxicity to reproduction of AAPBs from the results of an reliable oral developmental toxicity / teratogenicity study on rats at doses up to and including the guideline limit dose of 1000 mg a. i./kg bw/day and reliable oral subchronic and subacute repeated dose toxicity studies with histopathological examination of the male and female reproductive organs (epididymides, testes, seminal vesicle, prostate, ovaries, fallopian tubes, uterus, vagina, mammary gland).

Therefore no classification is required for toxicity to reproduction according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Additional information