2-Propenoic acid, 2-methyl-, 7-oxabicyclo[4.1.0]hept-3-ylmethyl ester

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April - May 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

See 'Principles of method if other than guideline'

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Principles of method if other than guideline:

DEVIATION
The starting dose was selected to be 300 mg/kg body weight. No compound-related mortality was recorded for all animals of step 1. As only slight signs of toxicity were noted a further step at this dose level was omitted for animal welfare reason - however opposed to OECD Guideline 423 (Annex 2c).
Instead, the second step was performed at a dose of 2000 mg/kg body weight. No compound-related mortality was recorded for any animal of step 2.
Based on these results and according to the acute toxic class method regime, a third step was performed with animals at a dose of 2000 mg/kg body weight. As opposed to previous steps where female animals were used, confirmatory step 3 was performed with male animals.
Compound-related mortality was recorded for one animal of step 3. Based on these results and according to the acute toxic class method regime no further testing was required.

REASON
Further testing of 3 animals at 300 mg/kg body weight was omitted for animal welfare reason. As confirmed by the results at 2000 mg/kg body weight, these animals would not have had an effect on classification of the test item.
The use of male animals in confirmatory step no. 3 was chosen on the assumption that a genderspecific susceptibility might be obtained.
This deviation did not influence the quality or integrity of the present study.

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

{7-oxabicyclo[4.1.0]heptan-3-yl}methyl 2-methylprop-2-enoate

EC Number:

688-147-2

Cas Number:

82428-30-6

Molecular formula:

C11 H16 O3

IUPAC Name:

{7-oxabicyclo[4.1.0]heptan-3-yl}methyl 2-methylprop-2-enoate

Test material form:

liquid

Details on test material:

- Name: 3,4-Epoxycyclohexylmethyl methacrylate
- Chemical Name: 2-Propenoic acid, 2-methyl-7-oxabicyclo[4.1.0]hept-3-ylmethyl ester
- CAS No.: 82428-30-6
- Physical State / Colour: liquid / light yellow, transparent
- Density: 1.079 g/mL at 20 °C
- Molecular Weight: 196.2 g/mol

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS

- Species/strain: WISTAR rats Crl: WI(Han)
- Source: Charles River
- Sex: Step 1-2 female (non-pregnant and nulliparous), Step 3 male
- Number of animals: 3 per step
- Age at the beginning of the study: 8-12 weeks old
- Body weight on the day of administration:
Step 1: animals no.: 1 - 3: 150 - 159 g (females)
Step 2: animals no.: 4 - 6: 166 - 172 g (females)
Step 3: animals no.: 7 - 9: 200 - 256 g (males)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

The animals were marked for individual identification by tail painting.
Prior to the administration a detailed clinical observation was made of all animals.
Prior to the administration food was withheld from the test animals for 16 to 19 hours (access to water was permitted). Following the period of fasting the animals were weighed and the test itemwas administered. Food was provided again approximately 4 hours post dosing.

HOUSING & FEEDING

- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10 %
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 per hour
- Free access to Altromin 1324 maintenance diet for rats and mice (lot no. 1526)
- Free access to tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were kept in groups in IVC cages, type III H, polysulphone cages on Altromin saw fibre bedding (lot no. 131113)
- Certificates of food, water and bedding are filed at the test institute
- Adequate acclimatisation period (at least five days) under laboratory conditions

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Doses:

300 and 2000 mg/kg bw

No. of animals per sex per dose:

300 mg/kg: 3 females
2000 mg/kg: 3 females + 3 males

Control animals:

no

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

2 500 mg/kg bw

Mortality:

yes, 1 male (200 mg/kg bw dosing) euthanized for ethical reasons

Clinical signs:

other: The following clinical signs have been observed throughout the study: Spontaneous activity reduced (slightly to moderately), Bradykinesia, Piloerection (slight to moderate), Eyes closed or half closed, Salivation (slight to moderate), Wasp waist, Kyphosi

Gross pathology:

With the exception of acute injection of blood vessels in the abdominal region, which is due to the euthanasia injection, no specific gross pathological changes were recorded for any animal.

Any other information on results incl. tables

CLINICAL SIGNS PER STEP

STEP 1: 300 mg/kg bw, 3 females

0 minutes	nsf
60 minutes	Spontaneous activity slightly reduced, Piloerection (slight)
2 d - 15 d	nsf

STEP 2: 2000 mg/kg bw, 3 females

0 minutes	nsf
60 minutes	Spontaneous activity moderately reduced, Kyphosis, Piloerection (moderate), Eyes closed, Wasp waist
2 d - 15 d	nsf

STEP 3: 2000 mg/kg bw, 3 males

0 minutes	nsf
60 minutes	Spontaneous activity moderately reduced, Piloerection (moderate), Eyes closed, Wasp waist, Bradykinesia, Salivation moderate, Moving the bedding
2 d - 15 d	2 males nsf, 1 male euthanized for ethical reasons

LD50 CUT-OFF

Dose [mg/kg bw]	No. of animals	No. of intercurrent deaths	LD50 Cut-Off
2000	6	1	2500 mg/kg bw
300	3	0

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present study, a single oral application of the test item 3,4-Epoxycyclohexylmethyl methacrylate to female rats at a dose of 300 mg/kg body weight was associated with slight signs of toxicity but no mortality.
Under the conditions of the present study, a single oral application of the test item 3,4-Epoxycyclohexylmethyl methacrylate to rats at a dose of 2000 mg/kg body weight was associated with signs of toxicity and mortality, which were slight more prominently expressed in male compared to female animals.

The median lethal dose of 3,4-Epoxycyclohexylmethyl methacrylate after a single oral administration to female and male rats, observed over a period of 14 days is: LD50 cut-off (rat): 2500 mg/kg bw

In conformity with the criteria given in Annex VI to Commission Directive 2001/59/EC [7] the test item has no obligatory labelling requirement for toxicity.
According to Annex I of Regulation (EC) 1272/2008 [8] the test item 3,4-Epoxycyclohexylmethyl methacrylate has no obligatory labelling requirement for toxicity and is not classified.
According to GHS (Globally Harmonized Classification System) [9] the test item 3,4-Epoxycyclohexylmethyl methacrylate has obligatory labelling requirement for toxicity and is classified into Category 5.

Executive summary:

SUMMARY

One group, with three female WISTAR Crl: WI(Han) rats, was treated with the test item by oral gavage administration at a dosage of 300 mg/kg body weight. The test item was suspended with the vehicle corn oil at a concentration of 0.03 g/mL and administered at a dose volume of 10 mL/kg.

Two groups, one female and one male group each of three WISTAR Crl: WI(Han) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The test item was suspended with vehicle corn oil at a concentration of 0.2 g/mL and administered at a dose volume of 10 mL/kg.

All animals used in the study after their entrance at the test institute were allowed to acclimatise to the laboratory conditions for at least 5 days. The animals were observed on delivery, on inclusion in the study and before administration for mortality/morbidity and other clinical signs. All animals were examined for clinical signs several times on the day of dosing and once daily until the end of the observation period. Their body weights were recorded on day 1 (prior to the administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

Step	Sex / No.	Starting dose [mg/kg]	No. of animals	No. of intercurrent deaths
1	female / 1 -3	300	3	0
2	female / 4 -6	2000	3	0
3	male / 7 -9	2000	3	1

All female animals treated with the test item at a dose of 300 mg/kg and 2000 mg/kg survived until the end of the study showing test-item related signs of toxicity.

One male animal treated with the test item at a dose of 2000 mg/kg had to be sacrificed for ethical reasons on test day 1. All remaining male animals survived until the end of the study.

The most relevant clinical findings in the animals treated with the test item at a dose of 300 mg/kg bw were reduced spontaneous activity and piloerection. All symptoms recovered within the day of treatment.

The most relevant clinical findings in the animals treated with the test item at a dose of 2000 mg/kg bw were reduced spontaneous activity, bradykinesia, piloerection, eyes closed, eyes half closed, salivation, moving the bedding and kyphosis. All symptoms recovered within up to the 2nd day post-dose. The clinical symptoms were similar in both genders, however, slightly more prominently expressed in male compared to female animals.

Throughout the 14-day observation period, the body weight gain of the surviving animals was within the normal range of variation for this strain.

Macroscopic findings of surviving animals: At necropsy, no treatment-related macroscopic findings were observed in any animal of any step. Macroscopic findings of animals not having survived until the end of the observation period: At necropsy, no treatment-related macroscopic findings were observed in any animal of any step.