[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 October, 2001 to 18 June, 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Reference 2

Reference Type:

other: Biocides assessment report

Title:

Assessment Report: Coco alkyltrimethylammonium chloride, Product-type 8 (Wood preservative); Regulation (EU) No 528/2012 concerning the making available on the market and use of biocidal products, Evaluation of active substances.

Author:

ECHA (RMS: Italy)

Year:

2016

Bibliographic source:

Approval ID: 1370-08; Asset No: EU-0014037-0000; Rapporteur Member State: Italy

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Quaternary ammonium compounds, coco alkyltrimethyl, chlorides

EC Number:

263-038-9

EC Name:

Quaternary ammonium compounds, coco alkyltrimethyl, chlorides

Cas Number:

61789-18-2

Molecular formula:

C(n+3) H(2n+10) N Cl (n=8-18)

IUPAC Name:

Quaternary ammonium compounds, cocoalkyl trimethyl, chloride

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Dihydrogen oxide

Test material form:

liquid

Specific details on test material used for the study:

- Physical state: Pale straw coloured liquid
- Analytical purity: 35.5% coco alkyl trimethyl ammonium chloride, 64.5% water
- Lot/batch No.: RHO20010082
- Storage condition of test material: Room temperature, in the dark

Test animals

Species:

rat

Strain:

other: Sprague-Dawley, Crl:CD® (SD) IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River (UK) limited, Margate, Kent
- Age at study initiation: 6 wk
- Weight at study initiation: Males: 141-183 g, females: 132-161 g
- Acclimation period: 14 d

ENVIRONMENTAL CONDITIONS
- Temperature: 21±2 °C
- Humidity: 55±15%
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: mixed with diet

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Mean dietary admixture concentrations were within acceptable limits for the purpose of the study (Gas chromatography).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily in feed

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 100, 500 and 2,000 ppm (i.e., equivalent to 22, 113 and 273 mg/kg bw/day after correction for 35.5% purity); the highest dose of 2,000 ppm was reduced to 1,000 ppm from Day 29 onwards due to deterioration in health of treated animals at 2,000 ppm.
Basis:
nominal in diet

No. of animals per sex per dose:

10 males and 10 females per dose

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: None

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (the day before start of treatment) and weekly thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Weekly throughout the study

FOOD EFFICIENCY: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily for each cage group

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before start of treatment and before termination of treatment (during week 12)
- Dose groups that were examined: All

HAEMATOLOGY: Yes
- Time schedule for collection of blood: End of treatment (Day 90)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: End of treatment (Day 90)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 0 (the day before start of treatment) and weekly thereafter
- Dose groups that were examined: All
- Battery of functions tested: Sensory activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes, Macroscopic lesions, Adrenals, Aorta, Bone and bone marrow (femur including stifle joint), Bone and bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Eyes, Gross lesions, Heart, ileum, Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (cervical and mesenteric), Mammary gland, Muscle (skeletal), Oesophagus, Ovaries, Pancreas, Pituitary gland, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles, Skin (hind limb), Spinal cord (cervical), Spleen, Stomach, Testes, Thymus, Thyroid/parathyroid, Tongue, Trachea, Urinary bladder, Uterus.

Statistics:

Data were processed to give group mean values and standard deviation where appropriate. Haematological, blood chemical, organ weight, weekly body weight gain and quantitative functional performance and sensory reactivity data were assessed for control and test substance treatment groups for dose response relationship by linear regression analysis, followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett's test. Where Levene's test showed unequal variances, the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney 'U' test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

effects observed, treatment-related

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

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

CLINICAL SIGNS AND MORTALITY: No mortality occurred during the study period.
Clinical signs - High dose animals developed clinical signs of toxicity from Day 7 onwards. These included hunched posture, pilo-erection, tiptoe gait, diarrhoea and red/brown staining of external body surface. Due to these effects, the dose level was reduced to 1000 ppm from Day 29 onwards. Clinical signs persisted following the reduction in dose level and included two incidents of pallor of extremities together with generalised fur loss. No clinically observable signs of toxicity were detected at the mid and the high doses.

Behavioural assessment: Detailed open-field observations conducted during the study confirmed the clinical signs of hunched posture, pilo-erection and tiptoe gait detected in high dose animals. No such effects were detected at the mid or low dose levels.

Functional performance test: No treatment-related changes were detected.

Sensory Reactivity Assessments: High dose females showed an increase in startle reflex parameters compared with controls. No such effects were detected for high dose males or for animals of either sex treated with the lower doses.

BODY WEIGHT AND WEIGHT GAIN: Reduced body weight gain was detected for high dose animals of either sex during the first five weeks of the study compared with controls. Mid dose males were similarly affected but this was confined to week 1 and 2 only. Body weight gain recovered following reduction in the dose level and was comparable with controls thereafter but terminal bodyweights for high dose animals and mid dose males remained lower than controls. No adverse effect on bodyweight gain was detected for 500 ppm females or for animals of either sex treated with the low dose.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Reduced food intake was observed in the high and mid dose animals throughout the study period compared with controls.

FOOD EFFICIENCY: Food efficiency was reduced over the first three weeks of the study but this was confined to the high dose group only. No adverse effect on dietary intake or food efficiency was detected at the low dose.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No appreciable intergroup differences were detected. High dose animals showed a reduced water intake on Day 6 of the study which recovered thereafter.

OPHTHALMOSCOPIC EXAMINATION: No treatment-related effects.

HAEMATOLOGY: No treatment-related effects.

CLINICAL CHEMISTRY: No treatment-related effects.

ORGAN WEIGHTS: No toxicologically important organ weight changes were detected. The reductions in absolute weight (including heart, kidneys, liver and thymus weight at the high dose and heart weight at the mid dose) and increases in relative weight (including high dose brain epididymides, kidneys, spleen, testes and ovaries weight), were all considered to be a result of reduced bodyweight development rather than test substance toxicity.

GROSS PATHOLOGY: No treatment-related macroscopic abnormalities.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related changes were observed in the spleen and kidneys. Lower severities of pigment accumulation were observed in the spleen of high dose male rats but not for the females (p <0.05). A higher incidence of pigment accumulation was observed in the kidneys of the high and mid dose male rats. In both tissues the pigment reacted positively to Perl's staining technique and was considered to be haemosiderin.
Haemosiderin is normally observed in kidneys of aged rats, where it accumulates in tubular epithelium. There are no indications of bleeding (black faeces, increase spleen weight, accelerated erythropoiesis).

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 40 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Clinical signs of toxicity, reduced body weight gain, reduced food efficiency and occurence of haemosiderine in kidneys of high dose animals.

Remarks on result:

other: reduction of body weight and food consumption can be considered secondary compared to the corrosive properties of the test substance; Haemosiderin is normally observed in kidneys of aged rats, where it accumulates in tubular epithelium.

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

The high dose showed clear effects related to palatability (esp. low food intake first week, clear increase after lowering dose level, lower BW gain), and clinical signs of discomfort starting after about 2 weeks leading to hunched posture of all animals, and red/brown staining of fur and piloerection in most day. This improved a little with lowering of dose. No specific toxicity was observed in haematology and clinical chemistry, terminal necropsy (besides stained fur in half of the animals and irritation stomach in one female), and organ weight (high dose are affected in a pattern consistent with lower BW, not considered indicative for organ toxicity). Histopathology only reported possible treatment related effects of haemosiderin accumulation in spleen and kidney. Generally, increased deposition of haemosiderin is followed from haemolytic effects.

Typically in cases of haemolytic anaemia higher grades of severity for both splenic extramedullary haemopoiesis and for splenic haemosiderin accumulation would be expected and this is often associated with accumulations of haemosiderin in the renal tubules. However, haemosiderin does not usually appear in excess in the renal tubules until a threshold increase in the spleen has been exceeded.

In this case, there was no evidence of increased haemolysis from the blood haematology parameters, and also no indication for an increased extramedullary haemopoiesis. Additionally, there was no Kupffer cell hemosiderin pigmentation of the liver. Haemosiderin is normally observed in kidneys of aged rats, where it accumulates in tubular epithelium. However, there are no indications of bleeding (black faeces, increase spleen weight, accelerated erythropoiesis). Other study parameters essentially did not show treatment-related effects. Also, as far as it is known, for haemosiderin deposition no significant difference is generally seen between males and females.

So in this case, there was no indication of extramedullary haemopoiesis, no effects in blood haematology, and if anything, the haemosiderin deposition seemed to decrease with dose in both males and females. In view that all reported levels are only minimal or incidental slight, levels in the males remain below levels in female, and normally there is no difference between male and females, it can be concluded that the reported statistical difference between control and dosed groups in the males are of no toxicological relevance. Therefore, in line with the study authors the NOAEL can be established at 500 ppm (i.e., equivalent to 40 mg a.i./kg bw/day).

Applicant's summary and conclusion

Conclusions:

Under the study conditions, based on effects on body weight, food efficiency and clinical signs, the NOAEL was established at 113 mg/kg bw/day (i.e., equivalent to 40.3 mg ai./kg bw/day).

Executive summary:

A 90-day study was conducted to determine the oral repeated dose toxicity of the test substance, Coco TMAC (active ingredient 35.5%), according to OECD Guideline 408 and EU Method B.26, in compliance with GLP. Sprague-Dawley rats were administered the test substance at concentrations of 0, 100, 500 or 2000 ppm (i.e., corresponding to 0, 22, 113 and 273 mg/kg bw/day in males and 0, 25, 121, 297 mg/kg bw/day in females) in the diet for 90 d. The active ingredient dose equivalent were calculated to be 0, 7.9, 40.3 and 96.9 mg a.i./kg bw/day in males and 8.8, 42.9, 105.3 mg a.i./kg bw/day in females. The highest dose of 2000 ppm was reduced to 1000 ppm from Day 29 onwards due to deterioration in health of the test animals at 2000 ppm. At the highest dose, the treatment-related findings were clinical signs of toxicity, reduced body weight gain and food efficiency, organ weight changes and microscopic changes in the spleen and kidneys. At the mid dose, reduced body weight gain (males) and reduced food consumption, reduced absolute heart weight and higher incidence of haemosiderin accumulation in the kidneys of males was observed. No treatment-related effects were observed at the lowest dose. Based on the results of the study, dietary administration of the test substance to rats for a period of 90 d at levels up to 273 mg/kg bw/day resulted in toxicologically significant effects at the high dose and marginal effects at the next lower dose of 113 mg/kg bw/day (500 ppm). No such effects were demonstrated at the lowest dose of 22 mg/kg bw/day (100 ppm). The changes observed at the mid dose (500 ppm) were considered to be minor, isolated effects associated with the reduced palatability of the test substance and were considered not to represent an adverse health effect. Therefore, based on effects on body weight, food efficiency and clinical signs the study authors established the NOAEL at the mid dose level of 500 ppm (i.e., equivalent to 40.3 mg ai./kg bw/day) (Jones, 2002).