Registration Dossier

Administrative data

Description of key information

Overall, considering the repeated dose studies with the read across substances (e.g., Coco TMAC) as mentioned in the Biocides assessment report, the main critical effects associated with these substances were due to their corrosive properties. The systemic effects such as the reduction of body weight and food consumption were considered to be secondary compared to the corrosive properties of the substances. Therefore, in the absence of ‘true’ systemic effects and in line with the biocides assessment reports, the derivation of a systemic DNEL has been considered to be non-relevant and only a qualitative local risk assessment has been conducted for the test substance.

Key value for chemical safety assessment

Toxic effect type:
concentration-driven

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
see 'Principles of method if other than guideline'
Deviations:
no
Principles of method if other than guideline:
Groups of 10 male and 10 female rats received test substance by oral gavage at the dose levels of 0, 30, 100 and 300 mg/kg bw/day for 28d. In addition, 5 male and 5 female animals were included in control and high dose group (recovery group). Animals were observed for clinical signs of toxicity, body weight changes, food and water consumption, haematological and biochemical parameters. Animals were necropsied and analysed for any visible abnormalities. Several tissues/organs were subjected to histopathological investigations in control and high dose group. Ophthalmological examination was also performed.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily
Remarks:
Doses / Concentrations:
0, 30, 100 and 300 mg/kg bw/day
Basis: other: The test substance used was a 24-26% aqueous solution but it is not clear if the doses mentioned are corrected for undiluted test substance.
No. of animals per sex per dose:
10 per sex per dose in main groups; 5 per sex in recovery groups (vehicle and highest dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: To check the reversibility of the effects, if any
- Post-exposure recovery period in satellite groups: at least 27d
Observations and examinations performed and frequency:
Animals were observed for clinical signs of toxicity. Body weight, food and water consumption were recorded. Haematological and biochemical parameters were analysed. All animals were necropsied and checked for macroscopically visible abnormalities. Several tissues/organs were preserved and processed for histopathological investigations in control and high dose group. Additionally, eyes were examined with slit lamp microscope for any treatment-related abnormality.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Other examinations:
Animals in the recovery groups were observed for at least 27d after termination of the treatment.
Statistics:
Yes, no detail available in the summary.
Clinical signs:
no effects observed
Description (incidence and severity):
Symptoms of local irritation were observed in the high dose group during the last week of treatment.

Mortality:
no mortality observed
Description (incidence):
No deaths occurred throughout the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Total body weight gain was comparable to control in test groups.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean food consumption in all treated groups was comparable to the control.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Water consumption was significantly increased in all animals of the high dose group.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
The examination of the eyes by slit lamp microscope showed no treatment-related effects.

Haematological findings:
no effects observed
Description (incidence and severity):
The haematological examinations revealed no substance related variation from the control values.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The highly significant increase in ALT-activity in the high dose group. The observed increase of the ALT-activity is considered to be an isolated finding as no other parameter is correlated with this deviation. Therefore, this finding is interpreted as incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The absolute and relative organ weights such as spleen and adrenals showed some substance related changes in the males of high dose group.



Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopical examination of the organs displayed substance related effects such as oedema of the mucosa of the forestomach, thickening of the mucosa and indication of ulceration in the animals of high dose group. Some additional observations like hydronephrosis, hydrometra and discolouration of the thymus showed no dose dependence and were therefore considered to be spontaneous. The observed irritative effects at the mucosa of the forestomach have been disappeared in the male and female animals of the recovery group 27d after termination of the treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
The forestomach of the male and female animals of the high dose group showed effects indicating local irritation like inflammatory oedema of the submucosa, sporadic ulceration and acanthosis of the mucosa up to papillomatous hyperplasia. These observations were considered to be due to the irritating properties of test substance and were considered not to be symptoms of a systemic toxicity. The animals of the recovery group of high dose showed a complete and regular regeneration of the forestomach mucosa.
Histopathological findings: neoplastic:
not examined
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on increase in water consumption, changes in the absolute and relative weights of the adrenals and spleens (in males) without corresponding effects on haematology, clinical chemistry and histology
Remarks on result:
other: corrected to 25 mg a.i./kg bw/day (as it was unclear from the study report if it was already corrected for the undiluted test substance)
Key result
Critical effects observed:
no
Conclusions:
Based on the results of the read across study, the rat 28 d NOEL for systemic effects was considered to be at 100 mg/kg bw/day, which is corrected to 25 mg a.i./kg bw/day (assuming its not been done in the study report)
Executive summary:

A study was conducted to determine the oral repeated dose toxicity of the read across substance, C16 TMAC (24 -26% active in water), according to a method similar to OECD Guideline 407, in compliance with GLP. Groups of 10 male and female rats were administered 0, 30, 100 and 300 mg/kg bw/day of read across substance by oral gavage for 28 days. The read across substance used was a 24-26% aqueous solution but it is not clear if the doses mentioned were corrected. There were no treatment-related changes at the 30 and 100 mg/kg bw/day. In the high-dose group there was an increase in water consumption, changes in the absolute and relative weights of the adrenals and spleens (in males) without corresponding effects on haematology, clinical chemistry and histology. The forestomach of the high-dose group showed few microscopic changes, however animals in the high-dose recovery group showed a complete and regular regeneration of the forestomach mucosa. Hence, the forestomach effects were considered to be due to the irritating properties of the read across substance rather than symptoms of systemic toxicity. Under the study conditions, the rat 28 d NOEL for systemic effects was considered to be at 100 mg/kg bw/day, which is corrected to 25 mg a.i./kg bw/day (assuming its not been done in the study report) (Potokar, 1991). Based on the results of the read across study, similar effects and NOAEL can be expected for the test substance.

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 29 October, 2001 to 18 June, 2002
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Species:
rat
Strain:
other: Sprague-Dawley, Crl:CD® (SD) IGS BR
Sex:
male/female
Details on test animals 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
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
Remarks:
Doses / Concentrations: 0, 100, 500 and 2,000 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). 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 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
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.
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).
Key result
Dose descriptor:
NOAEL
Effect level:
113 mg/kg bw/day (actual dose received)
Based on:
test mat.
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.
Remarks:
equivalent to 40.3 mg ai./kg bw/day
Key result
Critical effects observed:
no

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).

Conclusions:
Based on the results of the read across study, based on effects on body weight, food efficiency and clinical signs, the NOAEL was established at 500 ppm or 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 read across substance, Coco TMAC (35.5% active in water), according to OECD Guideline 408 and EU Method B.26, in compliance with GLP. Sprague-Dawley rats were administered the read across 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 read across 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 read across 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. Under the study conditions, based on effects on body weight, food efficiency and clinical signs, the NOAEL was established at 500 ppm or 113 mg/kg bw/day (i.e., equivalent to 40.3 mg ai./kg bw/day) (Jones, 2002). Based on the results of the read across study, similar NOAELs are expected for the test substance.

Endpoint:
chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
1976
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
no guideline required
Principles of method if other than guideline:
The test substance is administered daily in graduated doses to several groups of experimental animals, normally for a period of 12 months. This duration is chosen to be sufficiently long to allow any effects of cumulative toxicity to become manifest, without the confounding effects of geriatric changes. The test chemical is normally administered by the oral route although testing by the inhalation or dermal route may also be appropriate. During the period of administration the animals are observed closely for signs of toxicity. Animals which die or are killed during the test are necropsied and, at the conclusion of the test, surviving animals are killed and necropsied.
GLP compliance:
not specified
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rats,: 3--4 weeks old, were distributed in 4 experimental groups of 10 males and 10 females each, balanced with respect to body weights
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: 3--4 weeks old
- Housing: he animals were housed in groups of 5 animals with free access to food and water
- Fasting period before study: free access to food and water
- Water (e.g. ad libitum): drinking water in concentrations calculated to deliver doses of approximately 10, 20, and 45 mg/kg/day


Route of administration:
oral: drinking water
Details on route of administration:
The rats were given test substance in drinking water in concentrations calculated to deliver doses of approximately 10, 20, and 45 mg/kg/day.
Vehicle:
water
Details on oral exposure:
The rats were given test substance in drinking water in concentrations calculated to deliver doses of approximately 10, 20, and 45 mg/kg/day.When the animals attained maturity (6-7 weeks on test) the concentrations of test substance in drinking water were 0.007, 0.014, and 0.032%.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
1 year
Frequency of treatment:
Daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
20 mg/kg bw/day (nominal)
Dose / conc.:
45 mg/kg bw/day (nominal)
No. of animals per sex per dose:
4 experimental groups of 10 males and 10 females each
Control animals:
yes
Details on study design:
Sprague-Dawley rats, 3--4 weeks old, were distributed in 4 experimental groups of 10 males and 10 females each, balanced with respect to body weights. The animals were housed in groups of 5 animals with free access to food and water. The rats were given test substance in drinking water in concentrations calculated to deliver doses of approximately 10, 20, and 45 mg/kg/day. Since adult rats consume less water in proportion to body weight than younger ones, the concentrations of test substance in drinking water were increased during the study so that the intake of surfactant was approximately constant throughout the experimental period. When the animals attained maturity (6-7 weeks on test) the concentrations of test substance in drinking water were 0.007, 0.014, and 0.032%.
The body weight, the length of the tail of each animal, and food and water consumption collectively for groups of 5 animals were recorded weekly during the first 2 months, biweekly during months 3 and 4 and monthly thereafter. Regular observations were made of the appearance and behaviour of
the animals. The animals were killed 56-60 weeks after the beginning of the experiment. Blood was collected by heart puncture. Differential counts, haemoglobin determinations, quantitative determinations of serum proteins, electrophoretic separation of serum proteins and determination of Na + and K + contents
in serum were carried out. Haemoglobin was determined spectrophotometrically, Na + and K + were measured using an atomic absorption spectrophotometer, serum proteins were determined by the biuret method and electrophoretic separation of serum proteins were performed on agarose gels. The liver, kidneys, heart, spleen, stomach, caecum, and submaxillary salivary glands were weighed. Specimens of the stomach and small intestine were fixed in buffered formalin, embedded in paraffin wax, and stained with Heidenhain's azan or kernechtrot.
Observations and examinations performed and frequency:
Observations:
Within 3 weeks after initiating the study, the body weights of both male and female rats drinking the water with the highest concentration of test substance (corresponding to 45 mg/kg/day) were significantly lower than those of the controls and other experimental groups. In males the body weight remained reducedthroughout the experimental period, but in females the body weight was significantly reduced only up to the 9th week of the study. Both males and females rceiving test substance

Examinations:
Blood was collected by heart puncture. Differential counts, haemoglobin determinations, quantitative determinations of serum proteins, electrophoretic separation of serum proteins and determination of Na + and K + contents in serum were carried out.
The liver, kidneys, heart, spleen, stomach, caecum, and submaxillary salivary glands were weighed. Specimens of the stomach and small intestine were fixed in buffered formalin, embedded in paraffin wax, and stained with Heidenhain's azan or kernechtrot.at a dose of 10 and 20 mg/kg/day had a somewhat higher body weight (not statistically significant) than controls at the end of the study. A growth rate exceeding that of controls in animals receiving sublethal doses of quaternary ammonium surfactants has previously been reported. In male rats receiving the highest dose of test substance a significantly decreased efficiency of food conversion, compared with controls, was found during the first 7 weeks of the study. In females the efficiency of food conversion did not differfrom that in control animals. Skeletal growth, as judged by the growth of the tail, was significantly reduced at the highest dose level in both males and females.The reduction in skeletal growth was parallel to the reduction in total body weight, indicating that test substance affected the growth of soft and hard tissues in the same manner.
Apart from the reduction in body weight, the only compound-related external sign of intoxication was wetting over the anterior ventral region which was often associated with a brown discolouration of the fur. It occurred only at the highest dose level and in about 50% of the animals. These signs decreased in intensity as the study progressed. Deaths were sporadic among the animals, being dependent on neither dose nor time, and apparently due to bacterial infections of the lungs.
Sacrifice and pathology:
Sacrifice:The animals were killed 56-60 weeks after the beginning of the experiment. Blood was collected by heart puncture

Pathology: No compound-related gross abnormalities were seen on autopsy and no microscopic alterations were found in the wall of the stomach and small intestine. There were a few cases in which the ratio of organ to body weight for treated groups differed significantly from controls. A reduced liver weight was found in males receiving the highest dose of test substance. Increased caecal weights were found in males at the two highest dose levels and in females at the highest. Distention and enlargement of caecum in animals fed quaternary ammonium surfactants have previously been reported.
The results of clinical laboratory studies revealed no significant alternations in haemoglobin content, differential counts, total serum proteins, electrophoretic pattern of serum proteins or in Na + and K + content in serum in treated animals as compared with controls. About 4 months after the beginning of the study urine
samples from at least 5 males and 5 females from each experimental group were tested for protein, glucose, and haemoglobin with reagent sticks. No significant
differences were found between treated animals and controls.
Statistics:
Student's t-test
Clinical signs:
effects observed, non-treatment-related
Mortality:
no mortality observed
Description (incidence):
The effects on growth and body weight were most likely caused by a lower nutrients supply, secondary to diarrhoea.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Within 3 weeks after initiating the study, the body weights of both male and female rats drinking the water with the highest concentration of test substance (corresponding to 45 mg/kg/day) were significantly lower than those of the controls and other experimental groups. In males the body weight remained reduced throughout the experimental period, but in females the body weight was significantly reduced only up to the 9th week of the study.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
In male rats of the high dose group, a significantly decreased efficiency of food conversion, when compared to that of controls, was found during the first 7 weeks. In females, the efficiency of food conversion did not differ from that in control animals.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
decreased efficiency of food conversion at high dose
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
A reduced liver weight was found in males receiving the highest dose of test substance. Increased caecal weights were found in males at the two highest dose levels and in females at the highest.
Gross pathological findings:
no effects observed
Details on results:
At 10 mg/kg bw/day: No effects
At 20 mg/kg bw/day: - Increased caecal weights were found in males at the two highest dose levels.
At 45 mg/kg bw/day:
- Body Weight: Within 3 weeks after initiating the study, the body weights of both male and female rats drinking the water with the highest concentration of test substance (corresponding to 45 mg/kg/day) were significantly lower than those of the controls and other experimental groups. In males the body weight remained reduced throughout the experimental period, but in females the body weight was significantly reduced only up to the 9th week of the study.
- Food conversion : In male rats receiving the highest dose of test substance a significantly decreased efficiency of food conversion, compared with controls, was found during the first 7 weeks of the study. In females the efficiency of food conversion did not differ from that in control animals.
- Skeletal growth: Skeletal growth, as judged by the growth of the tail, was significantly reduced at the highest dose level in both males and females.

The effects on growth and body weight development observed in the study are considered to be an indirect effect of the administration of test substance in the drinking water. Since the test substance caused diarrhoea which resulted in a faster passage of food through the gastrointestinal tract and reduced absorption of nutrients.

- Gross pathology : No compound-related gross abnormalities were seen on autopsy and no microscopic alterations were found in the wall of the stomach and small intestine
- Organ Weight: A reduced liver weight was found in males receiving the highest dose. Increased caecal weights were found in males at the two highest dose levels and in females at the highest.

The increased relative caecum weight may be due to proliferation of the endothelial cell layer caused by an increased replacement rate due to a faster flux of the intestinal contents.

- Pathology: No significant differences were found between treated animals and controls.


Key result
Dose descriptor:
NOAEL
Effect level:
45 mg/kg bw/day (nominal)
Sex:
male/female
Remarks on result:
other: The effects on body weight, growth, caecal weight are considered to be indirect effects secondary to the irritant nature of the test substance, which led to diarrhoea.
Key result
Dose descriptor:
NOEL
Effect level:
10 mg/kg bw/day (nominal)
Sex:
male/female

Apart from the reduction in body weight, the only compound-related external sign of intoxication was wetting over the anterior ventral region which was often associated with a brown discolouration of the fur. It occurred only at the highest dose level and in about 50% of the animals. These signs decreased in intensity as the study progressed. Deaths were sporadic among the animals, being dependent on neither dose nor time, and apparently due to bacterial infections of the lungs.

Conclusions:
Based on the results of the read across study, the NOAEL for the test substance was considered to be 45 mg/kg bw/day and NOEL for the test substance was considered to be 10 mg/kg bw/day.
Executive summary:

A 1-year study was conducted to determine repeated dose toxicity of the read across substance, C16 TMAB (99% active) in rats. Read across substance was given to the male and female Sprague-Dawley rats in the drinking water at concentrations of 0.007, 0.014 and 0.032 % i.e., equivalent to 10, 20 and 45 mg/kg bw/day. Male and female rats of the high dose groups showed a significantly reduced body weight development, decreased efficiency of food consumption, decreased skeletal growth. In both males and females, an increased relative caecum weight was found in the high (males and females) and mid (males only) dose groups. No compound related changes were observed in haematological and clinical-chemical analyses of blood and urine. No gross necropsy changes were seen, and no microscopic alterations were found in the wall of stomach and small intestine of treated rats. No other tissues were histopathologically examined. The effects on growth and body weight development observed in the study are considered to be an indirect effect of the administration of read across substance in the drinking water. Since the read across substance caused diarrhoea which resulted in a faster passage of food through the gastrointestinal tract and reduced absorption of nutrients. The increased relative caecum weight may be due to proliferation of the endothelial cell layer caused by an increased replacement rate due to a faster flux of the intestinal contents. This explanation is supported by the lack of histopathologic alterations of the small intestine. Under the study conditions, the NOAEL for the read across substance was considered to be 45 mg/kg bw/day and NOEL for the read across substance was considered to be 10 mg/kg bw/day (Isomma, 1976). Based on the results of the read across study, similar effects and NOAEL can be expected for the test substance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
40.3 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The information requirement for this tonnage band is sufficiently met with the available data.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Critical effects observed:
not specified
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
However, few deficiencies were observed in the study such as tested with lesser number of animals (i.e., 10/group rather than 20/group as per guideline) and histoptahology of limited organs was performed.
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure:
- % coverage: 25% of the body surface area.
- Time intervals for shavings or clipplings: all rabbits was abraded with a clipper head prior to the start of each application

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Following the exposure period, the treated skin surface was cleaned with water

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0 or 10 mg/kg bw/day
- Concentration (if solution): 0 or 0.5% aqueous solutions, respectively. The dosage volume was 2.0 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, The animals were restrained with collars during the exposure period
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6.5 to 7 hours
Frequency of treatment:
5 days/week for 4 wks
Remarks:
Doses / Concentrations: 0 or 10 mg/kg/day
Basis: no data
No. of animals per sex per dose:
5 New Zealand albino rabbits/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: All rabbits were examined daily for clinical signs and mortality. Dermal irritation readings were recorded daily. The animals were weighed weekly during the exposure period. Blood was collected for haematology measurements before initiation of dosing and prior to termination. Liver and kidneys were weighed at necropsy. A complete list of tissues
was collected for histopathological evaluation
- Frequency of observations and weighing: weekly
- Necropsy of survivors performed: yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: twice daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected for haematology measurements before initiation of dosing and prior to termination

OTHER:
Mortality : twice daily
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes Liver and kidneys were weighed at necropsy. A complete list of tissues was collected for histopathological evaluation
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Treated areas of the skin that showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
- Two control group animals died during the study.
- Slight to moderate erythema was observed in all treated rabbits between days 4 and 8, but disappeared in 4 rabbits by day 17. Very slight to slight oedema was observed between days 6 and 12 in 4 rabbits and subsided by day 17. Two rabbits had intermittent slight oedema during week 4, and one rabbit developed oedema on day 20. No evidence of desquamation or leather-like skin was present in these animals. In the other rabbits, slight atonia occurred up to week 4 in 3 animals. Slight skin fissuring was observed in most of the rabbits but typically disappeared by the end of the study. There were no treatment-related effects on body weight, haematology, organ weight, gross necropsy findings or histopathology, except for treated areas of the skin that showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
other: see 'Remark'
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for the test substance is considered to be 10 mg/kg bw/day.
Executive summary:

A 28-day study was conducted to determine the repeated dose dermal toxicity of the read across substance, C16 TMAC (54.5% active in aqueous isopropanol), in New Zealand albino rabbits (both sexes) according to a method similar to OECD Guideline 410. The purity was not specified and the study included a lower than recommended number of animals (i.e., 10/group rather than 20/group as per guideline) and histopathology was performed only on limited organs. The read across substance (0 and 10 mg/kg bw/day) was applied to the shaved, intact skin of groups of 5 New Zealand albino rabbits/sex/group for 6.5 to 7 h, 5 days/week for 4 weeks. Dermal irritation readings were recorded daily. The animals were weighed weekly during the exposure period. Blood was collected for haematology measurements before initiation of dosing and prior to termination. Liver and kidneys weights were recorded at necropsy and limited histopathology was conducted. There were no systemic treatment-related effects on body weights, haematology, organ weights, gross necropsy findings or histopathology. Treated areas of the skin showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate. Under the study conditions, the 28d NOAEL for male and female rabbits were determined to be 10 mg/kg bw/day (TRS-HPV, 2001). Based on the results of the read across study, similar absence of systemic effects can be expected for the test substance.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
10 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
Due to some deficiencies in the study, the 90-day oral study has been considered further for hazard and risk assessment.

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Study period:
1979
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
However, few deficiencies were observed in the study such as tested with lesser number of animals (i.e., 10/group rather than 20/group as per guideline) and histoptahology of limited organs was performed.
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)
GLP compliance:
not specified
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Type of coverage:
open
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure:
- % coverage: 25% of the body surface area.
- Time intervals for shavings or clipplings: all rabbits was abraded with a clipper head prior to the start of each application

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Following the exposure period, the treated skin surface was cleaned with water

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0 or 10 mg/kg bw/day
- Concentration (if solution): 0 or 0.5% aqueous solutions, respectively. The dosage volume was 2.0 mL/kg bw

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes, The animals were restrained with collars during the exposure period
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
6.5 to 7 hours
Frequency of treatment:
5 days/week for 4 wks
Remarks:
Doses / Concentrations: 0 or 10 mg/kg/day
Basis: no data
No. of animals per sex per dose:
5 New Zealand albino rabbits/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Duration of observation period following administration: All rabbits were examined daily for clinical signs and mortality. Dermal irritation readings were recorded daily. The animals were weighed weekly during the exposure period. Blood was collected for haematology measurements before initiation of dosing and prior to termination. Liver and kidneys were weighed at necropsy. A complete list of tissues
was collected for histopathological evaluation
- Frequency of observations and weighing: weekly
- Necropsy of survivors performed: yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes / No / No data
- Time schedule: twice daily

DERMAL IRRITATION (if dermal study): Yes
- Time schedule for examinations: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood was collected for haematology measurements before initiation of dosing and prior to termination

OTHER:
Mortality : twice daily
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes Liver and kidneys were weighed at necropsy. A complete list of tissues was collected for histopathological evaluation
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
Treated areas of the skin that showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
- Two control group animals died during the study.
- Slight to moderate erythema was observed in all treated rabbits between days 4 and 8, but disappeared in 4 rabbits by day 17. Very slight to slight oedema was observed between days 6 and 12 in 4 rabbits and subsided by day 17. Two rabbits had intermittent slight oedema during week 4, and one rabbit developed oedema on day 20. No evidence of desquamation or leather-like skin was present in these animals. In the other rabbits, slight atonia occurred up to week 4 in 3 animals. Slight skin fissuring was observed in most of the rabbits but typically disappeared by the end of the study. There were no treatment-related effects on body weight, haematology, organ weight, gross necropsy findings or histopathology, except for treated areas of the skin that showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate.
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
dermal irritation
other: see 'Remark'
Key result
Critical effects observed:
no
Conclusions:
Under the study conditions, the NOAEL for the test substance is considered to be 10 mg/kg bw/day.
Executive summary:

A 28-day study was conducted to determine the repeated dose dermal toxicity of the read across substance, C16 TMAC (54.5% active in aqueous isopropanol), in New Zealand albino rabbits (both sexes) according to a method similar to OECD Guideline 410. The purity was not specified and the study included a lower than recommended number of animals (i.e., 10/group rather than 20/group as per guideline) and histopathology was performed only on limited organs. The read across substance (0 and 10 mg/kg bw/day) was applied to the shaved, intact skin of groups of 5 New Zealand albino rabbits/sex/group for 6.5 to 7 h, 5 days/week for 4 weeks. Dermal irritation readings were recorded daily. The animals were weighed weekly during the exposure period. Blood was collected for haematology measurements before initiation of dosing and prior to termination. Liver and kidneys weights were recorded at necropsy and limited histopathology was conducted. There were no systemic treatment-related effects on body weights, haematology, organ weights, gross necropsy findings or histopathology. Treated areas of the skin showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate. Under the study conditions, the 28d NOAEL for male and female rabbits were determined to be 10 mg/kg bw/day (TRS-HPV, 2001). Based on the results of the read across study, similar absence of systemic effects can be expected for the test substance.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The test substance has been classified as corrosive (no threshold derived)

Additional information

Oral:

Study 1: A study was conducted to determine the oral repeated dose toxicity of the read across substance, C16 TMAC (24 -26% active in water), according to a method similar to OECD Guideline 407, in compliance with GLP. Groups of 10 male and female rats were administered 0, 30, 100 and 300 mg/kg bw/day of read across substance by oral gavage for 28 days. The read across substance used was a 24-26% aqueous solution but it is not clear if the doses mentioned were corrected. There were no treatment-related changes at the 30 and 100 mg/kg bw/day. In the high-dose group there was an increase in water consumption, changes in the absolute and relative weights of the adrenals and spleens (in males) without corresponding effects on haematology, clinical chemistry and histology. The forestomach of the high-dose group showed few microscopic changes, however animals in the high-dose recovery group showed a complete and regular regeneration of the forestomach mucosa. Hence, the forestomach effects were considered to be due to the irritating properties of the read across substance rather than symptoms of systemic toxicity. Under the study conditions, the rat 28 d NOEL for systemic effects was considered to be at 100 mg/kg bw/day, which is corrected to 25 mg a.i./kg bw/day (assuming its not been done in the study report) (Potokar, 1991). Based on the results of the read across study, similar effects and NOAEL can be expected for the test substance.

Study 2: A 90-day study was conducted to determine the oral repeated dose toxicity of the read across substance, Coco TMAC (active: 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 113 mg/kg bw/day or 40.3 mg ai./kg bw/day) (Jones, 2002).The accumulation of haemosiderin in the spleen and kidneys at high and mid doses can be considered questionable for their toxicological relevance as they were not associated with any signs of bleeding (black faeces, increased spleen weight, accelerated erythropoiesis) and occurred in the absence of any effects on haematological parameters. Also, their levels in the males remained below the levels in female, whereas typically there is no known difference between the sex of animals for this effect. Further, the reduction in body weight and body weight gain, was attributed to local gastrointestinal irritation/corrosion and consequent reduced food intake without observing any primary systemic effect.

As per the Biocides assessment report on Coco TMAC, which was published by the Italian authorities in April 2016, the NOAEL in the above study was reported to be at the lowest dose 100 ppm (i.e., corresponding to 22 mg/kg bw/day or 7.9 mg a.i./kg bw/day). This was based on reduced food consumption with consequent lower body weight gains over the study period (10%) and the occurrence of haemosiderine in kidneys a dose-dependence effect that was considered to be treatment-related at LOAEL (500 ppm). Only at levels when serious differences in body weight were more than 20% compared to the control, other clinical effects occured. Similar results were obtained from subchronic feeding studies with other read across substances didecyldimethylammonium chloride (DDAC; CAS: 7173-51-5) (NO(A)EL=42 mg/kg bw/day) and C12-16 ADBAC (NOAEL of 68 mg a.s./kg bw/day) in rats, where the critical effect upon oral exposures were decreased body weight gain. Results from 90-day dog studies with read across substances DDAC and C12-16 ADBAC, with corresponding NOAELs at 15 and 45 mg a.i./kg bw/day (i.e., 486 and 1250 ppm), do not indicate species differences towards mechanism of toxicity by quaternary ammonium compounds. Further, the two highest doses (10 and 20 mg/kg bw/day) in a 1-year oral gavage study in dogs for DDACresulted in g.i.-related complications including emesis and abnormal faeces, resulting in death of 2 out of 4 animals at 20 mg/kg bw/day. The irritation/corrosive properties linked clinical signs observed in all the animals treated at 10 mg/kg bw/day (emesis, salivation, soft/loose faeces) persisted for the entire study duration. Only small amounts of the test substance were considered to be systemically available without giving rise to any significant systemic effect except for 10-15% decrease in body weight, which were considered to be secondary to effects in the gut. The NOAEL for local effects on gut mucosa was fixed equal to 3 mg/kg bw/day, whereas the systemic NOAEL was at 10 mg/kg/day for DDAC. Based on the outcome of the effect assessment, it was concluded that the AEL cannot be regarded as a “true” systemic threshold and therefore AEL approach based risk assessment should not to be performed. Consequently only a semi-quantitative local risk assessment was considered for the active substance (ECHA biocides assessment report, 2016).

Study 3:

A 1-year study was conducted to determine repeated dose toxicity of the read across substance, C16 TMAB (99% active) in rats. Read across substance was given to the male and female Sprague-Dawley rats in the drinking water at concentrations of 0.007, 0.014 and 0.032 % i.e., equivalent to 10, 20 and 45 mg/kg bw/day. Male and female rats of the high dose groups showed a significantly reduced body weight development, decreased efficiency of food consumption, decreased skeletal growth. In both males and females, an increased relative caecum weight was found in the high (males and females) and mid (males only) dose groups. No compound related changes were observed in haematological and clinical-chemical analyses of blood and urine. No gross necropsy changes were seen, and no microscopic alterations were found in the wall of stomach and small intestine of treated rats. No other tissues were histopathologically examined. The effects on growth and body weight development observed in the study are considered to be an indirect effect of the administration of read across substance in the drinking water. Since the read across substance caused diarrhoea which resulted in a faster passage of food through the gastrointestinal tract and reduced absorption of nutrients. The increased relative caecum weight may be due to proliferation of the endothelial cell layer caused by an increased replacement rate due to a faster flux of the intestinal contents. This explanation is supported by the lack of histopathologic alterations of the small intestine. Under the study conditions, the NOAEL for the read across substance was considered to be 45 mg/kg bw/day and NOEL for the read across substance was considered to be 10 mg/kg bw/day (Isomma, 1976).Based on the results of the read across study, similar effects and NOAEL can be expected for the test substance.

Overall, considering the repeated dose studies with Coco TMAC or other read across substances as mentioned in the Biocides assessment report, the main critical effects associated with these substances were due to their corrosive properties. The systemic effects such as the reduction of body weight and food consumption were considered to be secondary compared to the corrosive properties of the substances. Therefore, in the absence of ‘true’ systemic effects and in line with the biocides assessment reports, the derivation of a systemic DNEL has been considered to be non-relevant and only a qualitative local risk assessment has been conducted for the test substance.

Dermal

A 28-day study was conducted to determine the repeated dose dermal toxicity of the read across substance, C16 TMAC (54.5% active in aqueous isopropanol), in New Zealand albino rabbits (both sexes) according to a method similar to OECD Guideline 410. The purity was not specified and the study included a lower than recommended number of animals (i.e., 10/group rather than 20/group as per guideline) and histopathology was performed only on limited organs. The read across substance (0 and 10 mg/kg bw/day) was applied to the shaved, intact skin of groups of 5 New Zealand albino rabbits/sex/group for 6.5 to 7 h, 5 days/week for 4 weeks. Dermal irritation readings were recorded daily. The animals were weighed weekly during the exposure period. Blood was collected for haematology measurements before initiation of dosing and prior to termination. Liver and kidneys weights were recorded at necropsy and limited histopathology was conducted. There were no systemic treatment-related effects on body weights, haematology, organ weights, gross necropsy findings or histopathology. Treated areas of the skin showed mild to marked acanthosis with active mitosis, hyperkeratosis, and partial to extensive necrosis of the epidermis and hair follicles, partly with encrustation and exudate. Under the study conditions, the 28d NOAEL for male and female rabbits were determined to be 10 mg/kg bw/day (TRS-HPV, 2001). Based on the results of the read across study, similar absence of systemic effects can be expected for the test substance.

Inhalation

The substance has a low vapour pressure (VP = 0.0058 Pa at 25°C), which is below the cut-off of 0.01 Pa set for defining low volatility substances, as per the ECHA Guidance R.7a. Therefore, due to the low VP, the test substance is unlikely that it will form inhalable dust, mist or fumes when handled and used in solid form. In case inhalable forms of the substance (either pure or in aqueous solutions) are created under particular conditions (e.g., spraying, elevated temperature/pressure), appropriate risk management measures (due to corrosive nature of the test substance) such as closed systems, exhaust ventilation or wearing of respirators are implemented to control exposure. Under such conditions, the risk to humans following inhalation exposure can be considered minimal and further testing involving vertebrate animals may be omitted, in accordance with Annex XI (1.2) of the REACH regulation. Nevertheless, a qualitative risk assessment has been carried out for this route, due to the corrosive nature of the test substance and the fact that the available repeated dose oral studies with the read across substance did not show any primary systemic effects; all observed effects were attributed to local gastrointestinal irritation/corrosion and consequent reduced food intake.

Justification for classification or non-classification

Based on the results from the read across oral and dermal repeated dose studies, the test substance does not warrant a classification for STOT RE according to the EU CLP criteria (Regulation 1272/2008/EC).