2,3-epoxypropyltrimethylammonium chloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

other: Assessment report

Adequacy of study:

other information

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Although the EU risk assessment report is secondary literature, all data and risk assessment for the human, health and the environment have been evaluated and reviewed by Finland prior to publication. The risk assessment report has been submitted to final approval and published in the Official Journal of the European Union C157/10 dated on 21.06.2008. Thus, it is considered the information reported are reliable with the restrictions that reliability of the data presented has not been assessed again.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

publication

Title:

European Union Risk Assessment 2,3-epoxypropyltrimethylammonium chloride CAS RN 3033-77-0 Einecs No: 221-221-0

Author:

EC

Year:

2008

Bibliographic source:

Risk Assessment. Final approved version. Rapporteur: Finland (FIN). European communities. Printed in Italy. 147pp

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

: no details on test animals, environmental conditions, analytical verification of doses, oral exposure, study design and urinalysis results.

Principles of method if other than guideline:

Not applicable.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,3-epoxypropyltrimethylammonium chloride

EC Number:

221-221-0

EC Name:

2,3-epoxypropyltrimethylammonium chloride

Cas Number:

3033-77-0

Molecular formula:

C6H14NO.Cl

IUPAC Name:

N,N,N-trimethyl(oxiran-2-yl)methanaminium chloride

Details on test material:

- Physical state: aqueous solution
- Analytical purity: 72.6 % test substance in aqueous solution
- Impurities (identity and concentrations): The original solution had about 10% impurities

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: no data
- Fasting period before study: no data
- Housing: no data
- Diet (e.g. ad libitum): no data
- Water (e.g. ad libitum): no data
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): no data
- Humidity (%): no data
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): no data

IN-LIFE DATE: no data

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

No data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

No data

Duration of treatment / exposure:

28 days

Frequency of treatment:

1 time daily/7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 3.16, 10.0, 31.6 or 100 mg/kg EPTAC (72.6%) bw/day
Basis:
actual ingested

No. of animals per sex per dose:

- The control and high dose group had 10 males and 10 females (Five high dose and five control group animals were submitted to a 4-week post-exposure observation period)
- The low- and mid-dose groups had five rats of each sex.

Control animals:

yes, concurrent vehicle

Details on study design:

No data

Positive control:

No data

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes

DETAILED CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

OPHTHALMOSCOPIC EXAMINATION: No data

HAEMATOLOGY: Yes
Haematology measurement was that recommended in the guideline and it was determined at 4 and 8 weeks.

CLINICAL CHEMISTRY: Yes
Clinical chemistry measurement was that recommended in the guideline and it was determined at 4 and 8 weeks.

URINALYSIS: Yes
Urinalysis was performed in weeks 4 and 8 (recovery groups)

NEUROBEHAVIOURAL EXAMINATION: No data

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Samples for histopathological evaluation were prepared of adrenal glands, bone marrow from sternum, bone marrow smear, brain, various sections of the intestine, heart, kidneys, liver, lungs, ovaries, spleen, stomach, ovaries, testes and thymus.

Other examinations:

No data

Statistics:

Yes, But not detailed.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
Most high dose animals exhibited in their clinical picture piloerection and sunken sides. Two females and one male showed disturbed general
condition before death or decreased muscle tone. In the high dose group, four females and one male died during the last week of administration. One female was sacrificed moribund at day 16 of treatment.

BODY WEIGHT AND WEIGHT GAIN
In the 31.6 and 100 mg/kg groups, the bodyweight development slowed in parallel with the changes in food consumption. The body weight gain in
the 31.6 and 100 mg/kg dose groups was significantly reduced during weeks 2-4 of the treatment period. The body weight in the 100 mg/kg group males at day 28 was about 45% lower than the control group and in the 31.6 mg/kg group at that time the mean weight was about 18% lower and in
the 10 mg/kg group a 12 % reduction was noted. The 100 mg/kg dose females had about 38 % lower mean body weight than the control.
No significant difference was noted in other groups or in the recovery group. After the recovery period, the male 100 mg/kg recovery group had
still about 29% lower mean body weight, when compared to control.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption decreased in the mid- and high-dose groups.
The food consumption dropped to less than 50 % of the controls within two weeks in high dose females and within four weeks in high dose males.
Unlike the female rats, who had an almost complete recovery in their food consumption one week after the end of administration, the male recovery
returned to normal after seven weeks.

HAEMATOLOGY
both sexes of the 100 mg/kg group had statistically significantly increased red blood cell count (RBCM100: 8%; RBCF100: 11%), haemoglobin content (HbM100: 8%; HbF100: 11%) and relative and absolute monocyte count (MonMctrl: 3, MonM%100: 5; MonFctrl: 2, MonF100: 6). In the males of the 100 mg/kg group, white blood cell count (-32%) and relative (%LymRELCtrlM: 90, %LymREL100M: 80) and absolute (LymABSCtrlM: 11.16, LymREL100M: 6.84) lymphocyte numbers decreased. Males of the 100 mg/kg group had statistically significantly increased number of segmented neutrophils
(%NeuSEGCtrlM: 6, %NeuSEG100M: 15). Females additionally exhibited statistically significantly increased haematocrit (15%) and relative lymphocyte
counts (%LymRELCtrlF: 92, %LymREL100F: 80) and a decreased platelet count (-14%). The changes in the neutrophile values persisted in males until
the end of the recovery period. After the recovery period males exhibited slightly reduced values for erythrocytes, haematocrit, haemoglobin,
leucocytes and lymphocytes. In the females of the recovery group, only RBC and Hb figures were significantly reduced.

CLINICAL CHEMISTRY
The males and females of the high dose group had an increase (M: 28%, F: 63%)) in the activity of aspartate aminotransferase (ASAT) and gammaglut-amyltransferase. The males and females of the high dose group also had statistically significantly increased (M: 117 %, F: 667 %) gamma-glutamyltra-nsferase (GGT), and, only in the 10 (31%) and 31.6 (41%) mg/kg group males, an increased alanine aminotransferase (ALT). Male rats of the high
dose group had higher chlorine level (99 vs. 96 mmol/l). Alkaline phosphatase (M:-42 %, F:51 %), total protein (M:-9 %, F: 13) were reduced in both
sexes of the high dose group. Glucose (-14 %) and cholesterol (-35 %) values were reduced additionally in high dose males. Blood urea was
decreased in the high dose males (-28 %) and dose-dependently in all but the lowest dose females (-22 %, -30 % and -42 %). Only females
had a reduction in cholinesterase, which was significant in all but the 10 mg/kg dose group. Triglycerides in males of 31.6 mg/kg dose group (-46 %) and in males and females at 100 mg/kg (M:-38 %, F: -34 %) were decreased. Additionally, K+ and Ca2+ -levels of the high dose males (Ca2+ and K+: -7 %) and females (Ca2+: 15 %, K+: 8 %) were statistically significantly reduced. In males, the deviations in clinical chemistry the decrease in blood
urea and triglycerides did not return to normal after 8 weeks. In the female recovery group, total protein, blood urea, cholinesterase and albumin
levels were significantly reduced at week 8. The authors considered the changes in clinical chemistry parameters as adaptive to exposure and
decreased food intake.

URINALYSIS
No data

ORGAN WEIGHTS
In organ weight measurements, male testis (-35-40%) and brain weight (-10 %) were reduced in the high dose group and remained so in the recovery group at week 9. Also the brain to body weight ratio and brain to testis weight ratio were bigger in that group. As stated above, the body weight in
the high dose group was about 50% less than that of the control animals. The male absolute liver weight was slightly reduced in the 10 (19 %) mg/kg and 100 mg/kg (50 %) group with no change in the ratio to body weight. The only significant organ weight change that correlated with the
administrated dose was found in the heart (Males from low to high dose: -21 %, -26 %, 43 %). In females, the absolute weighs of liver (41 %) and heart (-38%) were decreased in the high dose group without a change in the ratio to body. Ovary weights were reduced in the 31.6 (~40 %) and 100 mg/kg (max 60 %) groups.

GROSS PATHOLOGY
Gross macroscopical examination revealed small spleen and thymus in both sexes of the high dose group than in other groups, which in microscopy was seen as a reduction of lymphatic tissue. The effect on thymus could not be adequately assessed in the intermediate dose groups,
as this organ was not taken at necropsy. The uterus of the two highest doses was reduced in size. However, the spleen, thymus or uterus weights
were not reported.

HISTOPATHOLOGY:
In the microscopic observations, the proximal convoluted tubule (PCT) of the kidneys had dose related necrosis and vacuolisation (lipid or fat deposits). In the 3.16 mg/kg dose group 3/5 males had minimal and 2/5 slight vacuolisation. All female rats of the 3.16 mg/kg group had minimal
vacuolisation. In the 10 mg/kg dose group, slight vacuolisation was present also in females (3/5). There was one case of minimal necrosis and hyperplasia, both in male rats of the 3.16 mg/kg group. One control male also showed tubular hyperplasia. In the 10 mg/kg group, proximal tubular
vacuolisation was minimal in 5/10 and slight in 5/10 animals, minimal necrosis and hyperplasia were present in 7/10 animals. Nuclear polyploidy
was present in 7/10 rats and 6/10 of those also had hyperplasia. The 31.6 mg/kg 7/10 animals of both sexes showed slight to 3/10 moderate
vacuolisation of the kidney proximal tubular cells, proximal tubular hyperplasia (minimal in 8/10, slight in 2/10), and polyploid nuclei in the proximal tubules and one case of an abnormal mitotic figure and reduced cellularity of the bone marrow. Minimal PCT-necrosis was present 9/10 animals
of this group. Minimal testicular atrophy was observed in 1/5 males and follicular atrophy of the ovaries was observed in 5/5 females (1/5 slight, 2/5 moderate, 4/5 marked). Persistent corpora lutea were reported in 4/5 females (2/5 moderate, 2/5 marked).
In the high dose group, vacuolisation was seen the proximal tubular cells of the kidney of inall animals and graded slight (1/10), moderate (5/10), marked (3/10) or massive (1/10). In the recovery group, the changes were slight in 5/10, moderate in 5/10 animals. Minimal PCT hyperplasia was
present in 6/10 animals of the dose group, two of the remaining animals had moderate to marked hyperplasia of the collective tubules and
transepithelial cells. In the recovery group PCT hyperplasia (minimal to moderate) was observed in 8/10 animals. Also minimal to moderate nuclear polyploidy was seen in 9/10 dose group and 9/10 recovery group animals. Minimal to moderate necrosis was present in the PCT epithelium of all
animals which was noted in the papilla of one male rat as marked.
Testicular atrophy was minimal in 1/5 males, moderate in 1/5 males and marked in 1/5 males of the dose group and minimal, slight or moderate in 1each of 5 recovery group males. Follicular atrophy of the ovaries was moderate in 2/5, marked in 1/5 and massive in 1/5 females of the dose group and slight in 1/5, marked in 3/5, and massive in 1/5 animals of the recovery group. Persistent corpora lutea were reported in 4/5 females of the
dose group (3/5 marked, 1/5 moderate) and 5/5 animals of the recovery group. Two of the recovery group females died after 14 and 16 days of
treatment.
In the spleen lymphoid atrophy (minimal 1/5, slight 1/5, moderate 1/5, marked 1/5) was observed in 5/5 males and 4/5 females (1/5 minimal, 1/5
slight, 2/5 marked) of the dose group and the 2/5 females that died premature in the recovery group. None of the animals that
survived the recovery period had lymphoid atrophy in the spleen.
The changes in the kidneys and the gonads persisted after the 4-week recovery period (100 mg/kg).
Again, only in the two highest doses, there was a reduction of all cell lines in the bone marrow, which correlated with the white blood cell reduction
seen in the haematology. The high dose animals showed occasional maturation disorders, vacuolisation and abnormal mitoses in the bone marrow
cells. The changes in the bone marrow, thymus or spleen were reversible after the recovery period.
Some animals of the high dose animals had mild focal hyper- and parakeratosis of the forestomach or mild erosions and haemorrhages in the
glandular stomach attributed to the local irritant property of EPTAC. The small intestine of these animals had villous atrophy and crypt necrosis. The changes in the stomach and small intestine were reversible.

Effect levels

1

Dose descriptor:

NOAEL

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects on the kidney proximal convoluted tubules (vacuolisation, necrosis, hyperplasia) already seen as minimal at 3.16 mg/kg bw/day

Remarks on result:

not determinable

Remarks:

no NOAEL identified

2

Dose descriptor:

LOAEL

Effect level:

3.16 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the effects on the kidney proximal convoluted tubules (vacuolisation, necrosis, hyperplasia) already seen at 3.16 mg/kg but evident at 10 mg/kg bw/day.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

No other information on results was available.

Applicant's summary and conclusion

Conclusions:

Under the test conditions of this study, based on the effects on the kidney proximal convoluted tubules (vacuolisation, necrosis, hyperplasia)
already seen as minimal at 3.16 mg/kg but evident at 10 mg/kg in the kidney a LOAEL of 3.16 mg/kg can be set. EPATC is therefore classified as STOT RE 2, H373 (May cause damage to organs through prolonged or repeated exposure ) according to the criteria of the CLP regulation ((No. 1272/2008 EC), and as Harmful: danger of serious damage to health by prolonged exposure if swallowed, (Xn, R48/22) according to the Directive 67/548/EEC.

Executive summary:

In a sub-acute study performed according to OECD 407 guideline and in compliance with GLP, 2,3 -epoxypropyltrimethylammonium (EPTAC), (72.6 % pure) diluted in water was administered by oral gavage to Wistar rats (10/sex/group for control and the high dose groups and 5/sex/group for low and mid dose groups) at 0, 3.16, 10.0, 31.6 or 100 mg/kg bw/day for four weeks. A control group received vehicle alone at the same volume-dosage. Five high dose and five control group animals were submitted to a 4-week post-exposure observation period

Under the conditions of this study, EPTAC is lethal to rats at dose of 100 mg/kg bw/day. Reduced food consumption, decreased in body weight gain are considered to be treatment related. The most sensitive organ appears to be the kidney in which minimal changes in morphology

could already be seen at an oral dose of 3.16 mg/kg bw/day. In the two highest dose groups, testes and ovaries showed focal atrophy that persisted through the recovery period. Moreover, the uterus was atrophic and morphologically in anoestrus in several of the female of these dose groups. Again in the high dose groups, red blood cells, haemoglobin and haematocrit values were slightly increased in both sexes. The high dose males also had increase in segmented neutrophils. These blood cell changes correlated with the findings of abnormal maturation and mitoses and vacuolisation seen in the bone marrow. The aminotransferases of the liver were increased during the treatment period in the high dose animals. However, these and most of the other clinical chemistry parameters were reversible after the recovery period. These changes may have been due to adaptive

processes of the liver.

Based on the effects on the kidney proximal convoluted tubules (vacuolisation, necrosis, hyperplasia) already seen as minimal at 3.16 mg/kg but evident at 10 mg/kg in the kidney a LOAEL of 3.16 mg/kg can be set. EPTAC is therefore classified as STOT RE 2, H373 (May cause damage to organs through prolonged or repeated exposure ) according to criteria of the CLP regulation ((No. 1272/2008 EC), and as Harmful: danger of serious damage to health by prolonged exposure if swallowed, (Xn, R48/22) according to the criteria of Directive 67/548/EEC.

This sub-acute toxicity study in the rats is acceptable and satisfies the guideline requirement for a sub-acute oral study (OECD 407) in rats.