Reaction mass of 6,6'-(2,2-Propanediyl)bis(3-phenyl-3,4-dihydro-2H-1,3-benzoxazine) and N-({4-[2-(3-phenyl-3,4-dihydro-2H-1,3-benzoxazin-6-yl)propan-2-yl]phenoxy}methyl)aniline

Endpoint summary

Description of key information

The test item did not induce toxic effects in the rat following an oral or a dermal administration at a level of 2000 mg/kg or following .

The lack of mortality demonstrates the LD50 is greater than 2000 mg/kg body weight for the acute oral toxicity and for acute dermal toxicity.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 February 2008 and Experimental completion date: 06 March 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: HanRcc: WIST (SPF)

Sex:

female

Details on test animals or test system and environmental conditions:

- Animals: Rat, HanRcc: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: RCC Ltd
- Laboratory Animal Services Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age when treated: 11 weeks
- Identification: Unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
- Randomization: Selected by hand at time of delivery. No computer generated randomization program.
- Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry

- Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with ranges for room temperature 22 ± 3 °C and for relative humidity between 30-70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.
- Accommodation: In groups of three in Makrolon type-4 cages with wire mesh tops and standard softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 77/07 (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) ad libitum.
- Water: Community tap water from Füllinsdorf ad libitum.

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Vehicle
- Identification: Corn oil
- Description: Yellowish oily liquid
- Batch number: 18787208
- Source: Carl Roth GmbH & Co. D-76185 Karlsruhe / Germany
- Stability of the Vehicle: Stable under storage conditions; expiration date: October, 2009

Dose Formulation
Dose levels are in terms of the test item as supplied by the sponsor.
The dose formulations were made shortly before each dosing occasion using a magnetic stirrer, a spatula and an Ultra-Turrax (Janke & Kunkel, D-79219 Staufen) as homogenizers.
The test item was first reduced into a fine powder using a mortar and a pestle. Thereafter, the test item was weighed into a tared glass beaker on a suitable precision balance and the vehicle was added (weight:volume).
Homogeneity of the test item in the vehicle was maintained during administration using a magnetic stirrer.

Treatment
The animals received a single dose of the test item by oral gavage administration at 2000 mg/kg body weight after being fasted for approximately 18 hours (access to water was permitted). Food was provided again approximately 3 hours after dosing.
The dosing volume was 10 mL/kg body weight.
Rationale: Oral administration was considered to be an appropriate application method as it is a possible route of human exposure during manufacture, handling and use of the test item.

Doses:

2000 mg/kg body weight

No. of animals per sex per dose:

3 females rats

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations and weighing:
- Mortality / Viability: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body Weights On test days 1 (prior to administration), 8 and 15. Clinical Signs Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15.
- Necropsy of survivors performed: yes, All animals were killed at the end of the observation period by Carbon dioxide asphyxiation and discarded after macroscopic examinations were performed. No organs or tissues were retained.
- Other examinations performed: clinical signs, body weight.

Statistics:

No statistical analysis was used.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred during the study.

Clinical signs:

No clinical signs were observed during the course of the study.

Body weight:

The body weight of the animals was within the range commonly recorded for this strain and age.

Gross pathology:

No macroscopic findings were recorded at necropsy.

Mortality/Clinical signs

Dose mg/kg bw

Animal N°

Sex

Signs

Test days

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

0.5*

1*

2*

3*

5*

2000

1

F

No clinical signs

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√

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√

√

√

√

√

√

√

√

√

√

√

√

√

2000

2

F

No clinical signs

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√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

2000

4

F

No clinical signs

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√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

2000

5

F

No clinical signs

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√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

2000

6

F

No clinical signs

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√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

√

Key:  √noted

* Examinations were performed within the first 30 minutes and 1, 2, 3 and 5 hours after treatment.

No clinical signs were evident in any animal during the acclimatization period.

Body weight

Dose mg/kg	Animal N°	Sex	Day 1 (treatment)	Day 8	Day 15
2000	1	F	177.2	188.7	203.2
2000	2	F	195.5	201.8	216.9
2000	3	F	177.9	189.8	197.7
2000	1	F	187.0	203.9	205.9
2000	2	F	188.1	206.5	215.1
2000	3	F	178.0	203.1	210.9

Body weight are presented in grams

Macroscopic Findings

Dose mg/kg bw	Animal N°	Sex	Mode of death	Findings
2000	1	F	S	No macroscopic findings
2000	2	F	S	No macroscopic findings
2000	3	F	S	No macroscopic findings
2000	1	F	S	No macroscopic findings
2000	2	F	S	No macroscopic findings
2000	3	F	S	No macroscopic findings

S: Scheduled necropsy

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The median lethal dose of the test item after single oral administration to female rats, observed over a period of 14 days is:
LD50 (female rat): greater than 2000 mg/kg body weight.

Executive summary:

The purpose of this study was to assess the acute toxicity of the test substance when administered by a single oral gavage to rats, followed by an observation period of 14 days.

Two groups, each of three female HanRcc:WIST (SPF) rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg body weight. The

test item was diluted in vehicle (corn oil) at a concentration of 0.2 g/mL and administered at a dosing volume of 10 mL/kg.

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded.

All animals were examined for clinical signs within the first 30 minutes and approximately 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

All animals survived until the end of the study period.

No clinical signs were observed during the course of the study.

The body weight of the animals was within the range commonly recorded for this strain and age.

No macroscopic findings were recorded at necropsy.

The median lethal dose of the test item after single oral administration to female rats, observed over a period of 14 days is:

LD50 (female rat): greater than 2000 mg/kg body weight

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Only this study is available and the Klimisch rate for this report is 1.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental starting date: 12 February 2008 and experimental compeltion date:04 March 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: HanRcc:WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
- Animals: Rat, HanRcc: WIST(SPF)
- Rationale: Recognized by international guidelines as a recommended test system.
- Breeder: RCC Ltd, Laboratory Animal Services, Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Number of Animals per Group: 5 males 5 females
- Age when treated: Males: 9 weeks
- Females: 11 weeks
- Identification: Unique cage number and corresponding color-coded spots on the tail. The animals were marked at acclimatization start.
- Randomization: Selected by hand at time of delivery.

Acclimatization: Under laboratory conditions, after health examination. Only animals without any visible signs of illness were used for the study.

Husbandry
- Room Number: 0105 / RCC Ltd, Füllinsdorf
- Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with ranges for room temperature 22 ± 3 °C and for relative humidity between 30-70 % (values above 70 % during cleaning process possible), automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.
Accommodation: During acclimatization in groups of five per sex in Makrolon type-4 cages with standard softwood bedding. Individually in Makrolon type-3 cages with standard softwood bedding ("Lignocel", Schill AG, CH-4132 Muttenz) during treatment and observation.

- Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet, batch no. 77/07 (Provimi Kliba AG, CH-4303 Kaiseraugst/Switzerland) ad libitum.
- Water: Community tap water from Füllinsdorf ad libitum.

Type of coverage:

semiocclusive

Vehicle:

corn oil

Details on dermal exposure:

One day before treatment, the backs of the animals were clipped with an electric clipper, exposing an area of approximately 10 % of the total body surface.
Only those animals without injury or irritation on the skin were used in the test.
On test day 1, the test item was applied at a dose of 2000 mg/kg body weight evenly on the intact skin with a syringe and covered with a semi-occlusive dressing. The dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.
- Application volume/kg body weight: 4 mL
Twenty-four hours after the application the dressing was removed and the skin was flushed with lukewarm tap water and dried with disposable paper towels. Thereafter, the reaction sites were assessed.
All animals were re-shaved on test day 8 to facilitate the reading of the local reactions.
Rationale: Dermal administration was used as this is one possible route of human exposure during manufacture, handling and use of the test item.

VEHICLE
- Identification: Corn oil
- Description: Yellowish oily liquid
- Batch number: 18787208
- Source: Carl Roth GmbH & Co. D-76185 Karlsruhe / Germany
- Stability of the Vehicle: Stable under storage conditions; expiration date: October, 2009
- Storage Conditions: At room temperature (range of 20 ± 5 °C), light protected.
- Safety Precautions: Routine hygienic procedures were used to ensure the health and safety of the personnel.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 males and 5 females

Control animals:

no

Details on study design:

- Duration of observation period following administration: 1 days
- Necropsy of survivors performed: yes. All animals were killed at the end of the observation period by Carbon dioxide asphyxiation and discarded after macroscopic examinations were performed. No organs or tissues were retained.
- Other examinations performed: clinical signs, body weight.
- Frequency of observations and weighing:

- Viability / Mortality: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15.
- Clinical Signs: Daily during the acclimatization period, during the first 30 minutes and at approximately 1, 2, 3 and 5 hours after administration on test day 1. Once daily during days 2-15. All abnormalities were recorded.
- Local Signs Once daily during days 2-15. All abnormalities were recorded.
- Body Weights: On test days 1 (prior to administration), 8 and 15.

Statistics:

No statistical analysis was used.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred during the study.

Clinical signs:

No clinical signs were observed during the course of the study.

Body weight:

The body weight of the animals was within the range commonly recorded for this strain and age.

Gross pathology:

No macroscopic findings were recorded at necropsy.

Other findings:

LOCAL SIGNS:

At removal of the application no local signs of irritation were observed in any animal. However, a yellowish staining produced by the test item was noted in all animals which persisted up to test day 15, the end of observation. A very slight erythema was recorded in one female on test day 8 and persisted up to test day 11. Slight scaling was recorded in all females from test day 8 to test days 13 and 15, respectively. Slight scabs were noted in two females from test day 10 to 13 or 15.

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The median lethal dose of the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is:
LD50 (rat): greater than 2000 mg/kg body weight

Executive summary:

Five male and five female HanRcc:WIST (SPF) rats were treated with the test substance at 2000 mg/kg by dermal application. The test item was diluted in vehicle (corn oil) at a concentration of 0.5 g/mL and administered at a volume dosage of 4 mL/kg. The application

period was 24 hours.

The animals were examined daily during the acclimatization period and mortality, viability and clinical signs were recorded. All animals were examined for clinical signs at approximately 30 minutes, 1, 2, 3 and 5 hours after treatment on day 1 and once daily during test days 2-5. Local signs were noted once daily from test day 2 to 15. Mortality/viability was recorded at approximately 30 minutes, 1, 2, 3 and 5 hours after administration on test day 1 (with the clinical signs) and twice daily during days 2-15. Body weights were recorded on day 1 (prior to administration) and on days 8 and 15. All animals were necropsied and examined macroscopically.

No deaths occurred during the study.

No clinical signs were observed during the course of the study.

At removal of the application no local signs of irritation were observed in any animal. However, a yellowish staining produced by the test item was noted in all animals which persisted up to test day 15, the end of observation. A very slight erythema was recorded in one female on test day 8 and persisted up to test day 11. Slight scaling was recorded in all females from test day 8 to test days 13 and 15, respectively. Slight scabs were noted in two females from test day 10 to 13 or 15.

The body weight of the animals was within the range commonly recorded for this strain and age.

No macroscopic findings were observed at necropsy.

The median lethal dose of the test item after single dermal administration to rats of both sexes, observed over a period of 14 days is: LD50 (rat): greater than 2000 mg/kg body weight

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

Only this study is available and the Klimisch rate for this report is 1.

Additional information

Two differents studies were performed to assess the acute oral toxicity and the acute dermal toxicity of the test item in the Wistar strain rat.

For the acute oral test, two groups, each of three female rats, were treated with the test item by oral gavage administration at a dosage of 2000 mg/kg diluted in corn oil and follwed by an observation period of 14 days.

- No mortality occured during the test, no abnormalities were noted at necropsy and all animals showed expected gains in body weight.

The median lethal dose of the test item after single oral administration to female rats, observed over a period of 14 days is: LD50 (female rat): greater than 2000 mg/kg body weight. (Globally Harminized Classification System-Unclassified).

In the acute dermal test, five males and five females was given a single, 24 hour, dermal application to the intact skin at a dose level of 2000 mg/kg body weight.

- No mortality occured during the test, no abnormalities were noted at necropsy 

-No signs of systemic toxicity were noted. 

- At removal of the application no local signs of irritation were observed in any animal. However, a yellowish staining produced by the test item was noted in all animals which persisted up to test day 15, the end of observation. A very slight erythema was recorded in one female on test day 8 and persisted up to test day 11. Slight scaling was recorded in all females from test day 8 to test days 13 and 15, respectively. Slight scabs were noted in two females from test day 10 to 13 or 15.

Regarding the body weight, it was within the range commonly recorded for this strain and age.

The acute dermal median lethal dose (LD₅₀) of the test item in the Wistar strain rat over a period of 14 days was found to be greater than 2000 mg/kg body weight.

Justification for classification or non-classification

Based on these results, the test item does not have to be classified and has no obligatory labelling requirement for acute oral and dermal toxicity according to the:

- Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007),

- Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.