C8-18 alkylampho(di)propionates

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 August 2012 - 15 February 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: EPA, Health Effects Test Guidelines OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, July 2000.

Deviations:

no

Principles of method if other than guideline:

In addition, the procedures described in the report essentially conform to the following guidelines:
- OECD Guidelines for Testing of Chemicals, Guideline 421, Reproduction/Developmental Toxicity Screening Test, July 1995.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test, July 2000.
- Commission regulation (EC) No 440/2008 Part B: Methods for the Determination of Toxicity and other Health Effects; B.7: "Repeated Dose (28 days) Toxicity (oral)". Official Journal of the European Union No. L142, May 2008.
- OECD Guidelines for Testing of Chemicals, Guideline 407, Repeated Dose 28-day Oral Toxicity Study in Rodents, October 2008.
- The United States EPA Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents, July 2000.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

other: aqueous solution

Details on test material:

- Physical state: Aqueous solution
- Appearance: Colourless to pale yellow liquid
- Composition of test material, percentage of components: see section confidential details on test material

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
Nulliparous and nonpregnant females and untreated animals were used at initiation of the study.
- Age at study initiation: Approximately 11 weeks.
- Weight at study initiation: weight at start of treatment was 305-341 gr (males) or 187-221 gr (females).
- Fasting period before study: no
- Housing:
Pre-mating: Animals were housed in groups of 5 animals/sex/cage in Macrolon cages.
Mating: Females were caged together with males on a one-to-one-basis in Macrolon cages.
Post-mating: Males were housed in their home cage with a maximum of 5 animals/cage. Females were individually housed in Macrolon cages. Pups were kept with the dam until termination
General: Sterilised sawdust as bedding material and paper as cage enrichment were supplied.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle.

IN-LIFE DATES
From: 22 August to 05 November 2012

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- Method of formulation: Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity (1.07) of the test substance. A correction was made for the purity/composition of the test substance. A correction factor of 38.56% was used. Dose levels were expressed as mg solid/kg body weight/day.
- Storage conditions of formulations: At ambient temperature.
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe.
- Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the treatment phase (02 October 2012), according to a validated method (Project 499453). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.
Results:
In the Group 1 formulation, no test substance was detected. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

Males were exposed for 29 days, i.e. 2 weeks prior to mating, during mating, and up to termination. Females were exposed for 41-54 days, i.e. during 2 weeks prior to mating, during mating, during post-coitum, and during at least 4 days of lactation. One female of Group 1, 2 females of Group 2, one female of Group 3 and 2 females of Group 4 were not dosed during littering.

Frequency of treatment:

Once daily, 7 d/w

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 mg solid/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
0, 259, 778, 2593 mg/kg bw/day based on test material (the substance)
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were based on results of a 10-Day dose range finding study in which 3 females/dose were dosed with 500 or 1000 mg solid/kg bw/d. No mortality or relevant clinical signs were noted. No effect on body weight, food consumption, macroscopy or liver/kidney weight were seen.
Based on the results of this range finding study, dose levels for the main study were: 100, 300 and 1000 mg solid/kg.

Since no toxicologically relevant clinical signs were observed in the range finding study, clinical observations in the main study were conducted immediately after dosing.

- Selection of animals for selected measurements: 5 animals/sex/group were randomly selected at allocation for functional observations, clinical pathology, macroscopic examination (full list), organ weights (full list) and histopathology. Only females with live offspring were selected.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS
- Time schedule: mortality/viability checked at least twice daily.

DETAILED CLINICAL OBSERVATIONS
- Time schedule: Daily from treatment onwards up to the day prior to necropsy, detailed clinical observations were made in all animals, after dosing (at no specific time point, but within a similar time period after dosing for the respective animals). Once prior to start of treatment and at weekly intervals during the treatment period this was also performed outside the home cage in a standard arena.

BODY WEIGHT
- Time schedule for examinations: Males and females were weighed on the first day of exposure and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum, and during lactation on Days 1 and 4.

FOOD CONSUMPTION
- Weekly, for males and females. Food consumption was not recorded during the mating period. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17 and 20 post-coitum and on Days 1 and 4 of lactation.

FOOD EFFICIENCY
No

WATER CONSUMPTION
No. Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION
No

HAEMATOLOGY
- Time schedule for collection of blood: at end of treatment.
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

CLINICAL CHEMISTRY
- Time schedule for collection of blood: at end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (with a maximum of 24 hours). Water was provided.
- How many animals: 5 animals/sex/group
- Parameters checked were: According to test guidelines

URINALYSIS
No

NEUROBEHAVIOURAL EXAMINATION
- Time schedule for examinations: The selected males were tested during Week 4 of treatment and the selected females were tested during lactation (all before blood sampling).
- Dose groups that were examined: all
- Battery of functions tested: According to test guidelines

Sacrifice and pathology:

GROSS PATHOLOGY
- All animals were fasted overnight (with a maximum of 24 hours) prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Selected 5 animals/sex/group and all animals that died spontaneously or were killed in extremis: According to test guidelines
- All remaining females which failed to deliver and the remaining males: According to test guidelines

ORGAN WEIGHTS
- Selected 5 animals/sex/group: According to test guidelines
- All remaining males: Epididymides and Testes

HISTOPATHOLOGY
- According to test guidelines

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 1; many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (Ref. 2; many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test (Ref. 3) was applied to frequency data.
- The following additional methods of statistical analysis were used: Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test (Ref. 4) to determine intergroup differences followed by the Wilcoxon test (Ref. 5) to compare the treated groups to the control group.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off
before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

References:
Ref. 1 Dunnett C.W., A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
Ref. 2 Miller R.G., Simultaneous Statistical Inference, Springer Verlag, New York (1981).
Ref. 3 Fisher R.A., Statistical Methods for Research Workers, Oliver and Boyd, Edinburgh (1950).
Ref. 4 Kruskal W.H. and Wallis W.A.. Use of ranks in one-criterion variance analysis. Journal of the American Statistical Association 47 (260): 583-621, December (1952).
Ref. 5 Wilcoxon, F. Individual comparisons by ranking methods. Biometrics, 1, 80-83 (1945).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Based on subjective appraisal.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

MORTALITY
No mortality occurred during the study period.

CLINICAL SIGNS
No clinical signs of toxicity were noted during the observation period.

Salivation seen after dosing among two mid dose animals and all high dose animals was considered to be a physiological response rather than a sign of systemic toxicity considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign may be related to irritancy/taste of the test substance. Also one control male showed salivation for one day.

Incidental findings that were noted included rales, scabs and alopecia. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological relevance.

FUNCTIONAL OBSERVATIONS
Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all selected animals.

The variation in motor activity did not indicate a relation with treatment.
All groups showed a similar habituation profile with very high activity in the first interval that decreased over the duration of the test period.

BODY WEIGHTS
No toxicologically relevant changes in body weights and body weight gain were noted.

For treated females, a statistically significantly increased body weight gain was noted on a few occasions. As it concerned a slight increase, all values were within normal limits and it was not consistent over time, these changes were not regarded toxicologically relevant.

FOOD CONSUMPTION
Food consumption before or after allowance for body weight was similar between treated and control animals.

HAEMATOLOGY
Haematological parameters of treated rats were considered not to have been affected by treatment.

The statistically significant changes noted for red blood cell distribution width (RDW) for all treated males were due to a high concurrent control value (due to two animals) and therefore not regarded treatment related.

The remaining statistically significant changes (prothrombin time for mid dose males and white blood cells for low dose females) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

CLINICAL BIOCHEMISTRY
At 1000 mg/kg, increased alanine aminotransferase (ALAT) concentrations were noted for both sexes; the increase was not considered toxicologically relevant as no corroborative findings were noted.

One male (no. 31) at 1000 mg/kg showed an increased value for aspartate aminotransferase (ASAT). As the remaining animals of this dose group showed a normal value, it was not considered treatment related but rather to have occurred by chance.

The remaining statistically significant changes (bile acids for high dose males and creatinine for low dose females) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain.

MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of treatment.

The incidence of incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included foci on the thymus, stomach or clitoral glands, nodule at the epididymides, discolouration of the mandibular lymph nodes, pelvic dilation of the kidneys, uterus containing fluid, enlarged spleen, and alopecia.

ORGAN WEIGHTS
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.

Relative liver weights were statistically significantly increased for females at 1000 mg/kg. However, as the increase was very slight, all values were within normal limits and no corroborative microscopic findings were noted, it was not considered toxicologically relevant.

The remaining statistically significant changes (thyroids for low dose males and prostate for mid dose males) were considered to be of no toxicological relevance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

MICROSCOPIC EXAMINATION
There was a slight increase in minor grades (minimal or slight) of hyaline droplets in cortical tubules of the kidneys in male rats. Minimal degree in two males at 100 mg/kg, minimal in two males at 300 mg/kg and at minimal or slight degree in three males at 1000 mg/kg. The hyaline droplets were considered to represent alpha2μglobulin, a normal protein in male rats which undergoes re-absorption in the proximal cortical tubules. A range of chemicals are known to increase hyaline droplet formation. This protein is not present in higher mammals, including man. The increase at 1000 mg/kg was not accompanied by degenerative tubular alterations.
In the thyroid glands, diffuse follicular hypertrophy/hyperplasia was noted at minimal degree in two control males, two males at 300 mg/kg and at minimal or slight in four males at 1000 mg/kg.
Neither of these findings achieved statistical significance. The grades recorded for both findings were within the range of background spontaneous alterations in this strain of rat and hence the increases noted may be regarded as adaptive in nature.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

Parental generation

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

other: solid content of the substance

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Remarks on result:

other: No toxicity was observed up to the highest dose level tested.

2

Dose descriptor:

NOAEL

Remarks:

Parental generation

Effect level:

2 593 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No toxicity was observed up to the highest dose level tested.

Remarks on result:

other: No toxicity was observed up to the highest dose level tested.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

In an oral OECD 422 screening study with rats, the parental NOAEL was determined to be 1000 mg solid/kg bw/day, based on the absence of toxicologically relevant adverse effects. The NOAEL based on the substance was determined to be 2593 mg/kg bw/day.

Executive summary:

In an oral OECD 422 screening study with rats, the parental NOAEL was determined to be 1000 mg solid/kg bw/day, based on the absence of significant adverse effects. The NOAEL based on the substance was determined to be 2593 mg/kg bw/day.