(carboxymethyl)dimethyl[3-[(1-oxoundecenyl)amino]propyl]ammonium hydroxide

Toxicity to reproduction

Administrative data

Endpoint:

toxicity to reproduction

Remarks:

other: Repeated Dose 28-Day Oral Toxicity in Rodents

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

2015-08-21 to 2015-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study, GLP

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral)) (2008)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) (adopted 3 October 2008)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

C8-10 Alkylamidopropyl betaine

IUPAC Name:

C8-10 Alkylamidopropyl betaine

Details on test material:

- Name of test material (as cited in study report): 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-(C8-10 acyl) derivs., hydroxides, inner salts
- Analytical purity: 34.65% active ingredient (betain)

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 27-29 days
- Weight at study initiation: 92-100 g for males and 80-95 g for females at arrival
- Fasting period before study: no
- Housing: up to 5 of one sex to a cage, in clear polisulphone solid bottomed cages; nesting material was provided inside suitable bedding bag and changed at least twice a week
- Diet (e.g. ad libitum): powdered laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: approx. 3 wk

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 55±15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: purified water (softened water by reverse osmosis)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was dissolved in the vehicle. The formulations were prepared daily at concentrations of 10, 30 and 50 mg/mL. Concentrations were calculated and expressed in terms of test item corrected against the declared purity.

VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Details on mating procedure:

animals were not mated

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration) and a 28 hour and 8 day stability at room temperature was verified in the same range, to confirm that the method was suitable and stability was satisfactory.
Final results for all levels were within the acceptability limits for concentration (90-110%).
Samples of the formulations prepared on Weeks 1 and 4 were analysed to check the concentration. Results of the analyses were within the acceptability
limits for concentration of solutions (90-110%)

Duration of treatment / exposure:

4 weeks + recovery period of 2 weeks

Frequency of treatment:

daily

Details on study schedule:

-animals were not mated

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 300, 500 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

5 male and 5 female; control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results with similar substances
- Rationale for animal assignment (if not random): computerised stratified randomisation to give approximately equal initial group mean body weights
- Rationale for selecting satellite groups: to assess recovery from any treatment-related effects
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

n.a.

Examinations

Parental animals: Observations and examinations:

MORTALITY: Yes
- twice daily

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily, approximately 1 hour after dosing

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once per week from the start of treatment
- parameters: gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern)

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly starting from the day of allocation to treatment group and just prior to necropsy

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OTHER: NEUROBEHAVIOURAL EXAMINATION, CLINICAL CHEMISTRY, HAEMATOLOGY
- described in more detail in section "Repeated dose toxicity"

Oestrous cyclicity (parental animals):

The histopathological evaluation of the oestrous cycle in the uterus/ cervix and vagina from all animals in the control and high dose groups of the
main phase was performed.

Sperm parameters (parental animals):

The testes and epididymides of main group animals were cut at 2-3 µm thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 4 weeks of treatment.

Litter observations:

n.a.

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Postmortem examinations (offspring):

n.a.

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test.
If the data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.

Reproductive indices:

n.a.

Offspring viability indices:

n.a.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

not examined

Details on results (P0)

CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study.
No clinical signs were seen during the study.
No toxicological relevant changes were observed at the weekly detailed clinical signs.
Statistically significant decreases in rearing were seen in males receiving 300 and 500 mg/kg bw/day during Weeks 3 and 4 of treatment, increases were seen
in females receiving 100 and 300 mg/kg bw/day during Week 3 and in females receiving 500 mg/kg bw/day during Week 4 of treatment, when compared to
controls. Since the direction of changes was opposite in the two sexes, the above mentioned changes were considered incidental and not treatmentrelated.

BODY WEIGHT AND WEIGHT GAIN
No differences in body weight were seen between treated and control animals during the study.

FOOD CONSUMPTION
No differences in food consumption were seen between treated and control groups during the study.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

ORGAN WEIGHTS
No relevant differences were seen in terminal body weights of treated animals respect to controls.
No toxicological relevance was attributed to the statistically significant increase in absolute (13%) and/or relative (9-10%) kidneys weight seen in females treated at 300 and/or 500 mg/kg/day not to the statistically significant increase in relative (up to 7%) kidneys weight seen in males treated at 300 and 500 mg/kg/day, when compared to controls, since these increases were slight and no changes were detected at the histopathological evaluation.
No other differences were seen in treated groups when compared to controls.

GROSS PATHOLOGY
No relevant changes were noted in treated animals. The sporadic changes observed in few treated animals could be considered incidental.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related changes were noted. All observed changes in organs and tissues are considered as incidental-age related findings, characteristically seen in untreated Sprague Dawley rats of the same age or comparable with control animals.

OTHER FINDINGS (PARENTAL ANIMALS)
- described in more detail in section "Repeated dose toxicity"

Effect levels (P0)

1

Dose descriptor:

NOEL

Remarks:

fertility parameters

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects up to and including the highest tested dose level

2

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

>= 500 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: no treatment-related adverse effects up to and including the highest tested dose level

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

n.a.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL for general toxicity and the NOEL for fertility parameters.

Executive summary:

In a subacute toxicity study according to OECD guideline 407 (2008) and EU method B.7 (2008) C8-10 Alkylamidopropyl betaine (34.65% a.i.) was administered to 5Hsd: Sprague Dawley SDrats/sex/dose in purified water by gavage at dose levels of 0 (control), 100, 300 and 500 mg/kg bw/day for 28 consecutive days. Control and high dose groups included 5 additional animals per sex to be sacrificed after 2 weeks of recovery.

No mortality occurred. No clinical signs and no changes were observed at the weekly detailed clinical observations. Neurotoxicity assessment did not reveal any treatment-related effects. No changes on body weight and food consumption were noted.

The lymphocytosis and monocytosis seen in single females dosed at 300 and/or 500 mg/kg bw/day showed reversibility at the end of the recovery period or comparability to control data. No toxicological relevant effects in coagulation and clinical chemistry parameters were observed. No differences were reported in terminal body weights and organ weights between treated and control animals and no treatment-related changes were noted at macroscopic and microscopic observations.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Normal physiology of the oestrous cycle (oestrous, metestrous, diestrous and proestrous) was noted in control and treated females. The morphological changes seen were normal when compared to each “oestrous phase” in the ovaries, uterus/cervix and vagina.

On the basis of the results obtained in this study, the dose level of 500 mg/kg bw/day was considered the NOAEL (No Observed Adverse Effect Level).

The NOEL for fertility parameters is 500 mg/kg bw/d.