Phenothiazine

Endpoint summary

Description of key information

Acute Toxicity: oral
- Standard acute method, pre-GLP and not according to OECD guideline
- Clinical observation: all animals had varying degrees of diarrhoea and were weak after administration
- LD50 = 1370 mg/kg body weight
Acute Toxicity: dermal
- OECD 402, conducted as limit test
- None of the animals died, no signs for systemic toxicity
- Signs of skin irritation were observed
- LD50 > 2000 mg/kg body weight

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:

Jan. 1977 to Feb. 1977

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Well performed study prior to GLP meeting scientific principles and similar to previous OECD TG.

Qualifier:

according to guideline

Guideline:

other: As outlined in "Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics" by the staff of the Division of Pharmacology, Food and Drug Administration, Department of Health, Education and Welfare, pages 17-25

Deviations:

not specified

GLP compliance:

no

Remarks:

Prior to GLP

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Albino rats (Sprague Dawley strain) obtained fromSasco, Inc., Omaha, Nebraska. (U.S.D.A. License No. 47-A-B-98).
- Animals were 40-50 days of age.
- Animals are examined upon receipt to check for any apparent abnormal condition.
- Any animal which did not appear to be normal was discarded.
- Animals were then transferred to individual cages in a temperature controlled room where they acclimated for seven days prior to initiation of the study.
- Animals werde maintained in individual cages throughout the study period.
- Purina Rat Chow and clean water was provided for each animal on an ad libitum basis throughout the study period except that they are fasted twenty-four hours prior to administration of the test compounds.

Route of administration:

oral: gavage

Vehicle:

cotton seed oil

Doses:

- Dose-range-findings study: 2000 - 3170 - 5024 - 7964 - 12623 - (1257 - 1992) mg/kg
- Main study: Five Dose Level Study (0.1 log intervals) 600 - 755 - 951 - 1197 - 1508 mg/kg

No. of animals per sex per dose:

1 male and 1 female in range-finding-study (1257 mg/kg in 1 male - 1992 mg/kg in 1 female)
5 males and 5 females per dose in main study

Control animals:

no

Details on study design:

Duration of observation period following administration:
- 14 days

Frequency of observations and weighing:
- Each animal was observed by trained technicians at approx. two hour intervals during the first working day following adminstration of the test compound.
- Thereafter, observations were made two or three times each day for the remainder of the study.
- Animals were observed for any visual sign of weakness, abnormal breathing or abnormal body movement.
- Further, if animals appeared unconscious, they were removed from their cage, checked for any abdominal distention, oral and anal areas were examined for any change and they were tested for response to stimulus (noise and prodding)

Statistics:

- Method of Litchfield-Wilcoxon was applied

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 370 mg/kg bw

95% CL:

> 1 223 - <= 1 534

Mortality:

Please see table "Mortalitiy Data of Main Study" in section "Remarks on results including tables and figures" below

Clinical signs:

other: Please see section "Remarks on results including tables and figures" below for details.

Gross pathology:

Data only on dose-range finding study, please see section "Remarks on results including tables and figures" below for details. No gross pathology performed in main study due to the results of the dose-range-finding-study.

Other findings:

Please see section "Remarks on results including tables and figures" below for details.

RESULTS OF MAIN STUDY

Animals in treatment groups 600, 755 and 951 mg/kg were slightly weak through the first eight hours following intubation. Animals in treatment groups 1197 and 1508 mg/kg were semi-conscious through the first eight hours. Animals in the lowest dosage groups which survived were weak through the first six or seven days of the study before returning to normal. Animals in the two highest dosage groups, which survived, remained weak throughout the fourteen day study period. All animals had varying degrees of diarrhoea.

Table: Mortality Data of Main Study

Rat No. and Sex	Dosage mg/kg	Mortality
5 male and 5 female	600	1 male found dead the morning of 6th study day
5 male and 5 female	755	1 male found dead the morning of 3rd study day
5 male and 5 female	951	1 female found dead the morning of 2nd study day
5 male and 5 female	1197	1 male found dead the morning of 3rd study day and  1 male found dead the morning of 4th study day
5 male and 5 female	1508	1 male and 1 female  found dead the morning of 2nd study day;  1 male and 1 female  found dead the morning of 3rd study day;  1 male and 1 female  found dead the morning of 4th study day

RESULTS OF DOSE-RANGE-FINDING-STUDY

Within one hour after intubation all animals were semi-conscious. Breathing appeared to be normal. Faecal material indicated the animals had some diarrhoea. Post mortem examination revealed all organs to be normal in size and color except for the stomach. The stomach was filled with the test material and the blood vessels of the intestinal tract appeared to be injected. All animals treated died, except for the single male rat which was tested at a dose of 1257 mg/kg body weight.

Interpretation of results:

harmful

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the data obtained and under the conditions this study was conducted, the LD50 of Phenothiazine Purified (Lot No. NB 36-145) is 1370 mg per kilogram body weight as calculated by Litchfield-Wilcoxon Statistical Method. The 95% confidence interval is 1223 to 1534 mg per Kg.

Executive summary:

Acute oral toxicity of phenothiazine was assessed in the present study. Animals in treatment groups 600, 755 and 951 mg/kg were slightly weak through the first eight hours following intubation. Animals in treatment groups 1197 and 1508 mg/kg were semi-conscious through the first eight hours. Animals in the lowest dosage groups which survived were weak through the first six or seven days of the study before returning to normal. Animals in the two highest dosage groups, which survived, remained weak throughout the fourteen day study period. All animals had varying degrees of diarrhoea.

Based on the data obtained and under the conditions this study was conducted, the LD50 of Phenothiazine Purified (Lot No. NB 36-145) is 1370 mg per kilogram body weight as calculated by Litchfield-Wilcoxon Statistical Method. The 95% confidence interval is 1223 to 1534 mg per kg. Classification according to 67/548/EEC phenothiazine is harmful, classification according to Regulation (EC) 1272/2008 would be Acute Toxicity Category 4 with H302.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 370 mg/kg bw

Acute toxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

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:

2010-03-17 to 2010-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well performed GLP study according to recent OECD guidelines.

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)

Remarks:

Certificate attached to full study report.

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: females: 18-19 weeks; males: 8-9 weeks
- Weight at study initiation: femailes: 215-228g; males: 240-260g
- Housing: Full barrier in an air-conditioned room, animals were kept individually in IVC cages, type III H, polysulphone cages on Altomin saw fibre bedding
- Diet (e.g. ad libitum): Altromin 1324, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Certificates of food, water and bedding are filed at the test facility
- Acclimation period: at least five days
- Female rats were nulliparous and non-pregnant

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12hrs / 12hrs

Type of coverage:

semiocclusive

Vehicle:

other: Corn oil (Sigma-Aldrich)

Details on dermal exposure:

TEST SITE
- Area of exposure: dorsal area of the trunk (approx. 24hrs before the test, the fur was removed by using an electrical clipper)
- % coverage: 10%
- Type of wrap if used: semi-occlusive dressing consisted of a gauze-dressing and non-irritating tape and was fixed with an additional dressing in a suitable manner.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): at the end of exposure period residual test item was removed by using corn oil
- Time after start of exposure: 24hrs

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): at a single dose of 2000 mg/kg body weight
- Constant volume or concentration used: yes
- For solids, paste formed: yes, moistened with vehicle
- The test item was ground to a fine powder in a mortar with the help of a pestle
- In order to ensure good skin contact it was moistened with the vehicle

VEHICLE
- Lot/batch no. (if required): lot no. 128K0040, expiry date: 25/04/2010
- Rationale: This vehicle was chosen due to its non-irritating characteristics

Duration of exposure:

24hrs

Doses:

2000mg/kg body weight

No. of animals per sex per dose:

5 females and 5 males

Control animals:

not required

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: weighing on day 1 (prior to application) and on days 8 and 15, clinical examination was made several times on day of dosing (at least once during the first 30mins and with special attention given during the first 4hrs post-dose).
- Necropsy of survivors performed: yes, all gross pathological changes were recorded.
- Other examinations performed: clinical signs, body weight

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: The test item showed no sign of acute dermal toxicity.

Mortality:

No mortalities.

Clinical signs:

other: No findings during the entire observation period.

Gross pathology:

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

Other findings:

Signs of irritation
- Erythema grade 1 was observed in 4/5 female and 5/5 male animals
- Erythema grade 2 was observed in 3/5 male animals
- Desquamation and eschar were observed in 5/5 female and 5/5 male animals
- Scratches were observed in 3/5 female and 5/5 male animals
- Not all signs of irritation were reversible with the observation period

TABLE: Skin Irritation – Individual Data – Females

Day after Start of Application	Animal No. 11		Animal No. 12		Animal No. 13		Animal No. 14		Animal No. 15
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	1/0	nf	1/0	nf	0/0	nf	0/0	*	0/0	*
day 3	1/0	nf	1/0	nf	0/0	nf	0/0	*	0/0	*
day 4	1/0	slight s	1/0	nf	0/0	nf	0/0	*	0/0	*
day 5	0/0	s, d	1/0	d	1/0	d	0/0	slight d	0/0	d
day 6	0/0	d, es	1/0	d	1/0	d, es	0/0	slight d	1/0	d, es
day 7	0/0	d, es	0/0	d, es	0/0	d, es	0/0	d, slight s	0/0	d, s
day 8	0/0	d, es	0/0	d, es	0/0	d, es	0/0	d	0/0	slight d
day 9	0/0	slight d, slight es	0/0	d, es	0/0	d, es	0/0	d	0/0	slight d
day 10	0/0	nf	0/0	d, es	0/0	d, es	0/0	moderate d	0/0	slight d
day 11	0/0	slight d, slight s	0/0	moderate d	0/0	slight d	0/0	slight d	0/0	nf
day 12	0/0	slight d	0/0	slight d	0/0	nf	0/0	nf	0/0	nf
day 13	0/0	slight d	0/0	slight d	0/0	nf	0/0	nf	0/0	nf
day 14	0/0	slight d	0/0	nf	0/0	nf	0/0	nf	0/0	nf
day 15	0/0	nf	0/0	nf	0/0	nf	0/0	nf	0/0	nf
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) d = desquamation; es = eschar; s = scratches; nf = no findings * orange discoloration at the application site, caused by the test item

 

TABLE: Skin irritation – Individual Data – Males

Day after Start of Application	Animal No. 21		Animal No. 22		Animal No. 23		Animal No. 24		Animal No. 25
	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments	E/O	Comments
day 2	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 3	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 4	0/0	*	0/0	*	0/0	*	0/0	*	0/0	*
day 5	2/0	d, es	1/0	d, es	1/0	d, es	2/0	d, es	2/0	d, es
day 6	1/0	d, es	1/0	d, es	0/0	d, es	1/0	d, es	1/0	d, es
day 7	0/0	d, es	0/0	d, es	0/0	d, es, **	0/0	d, es, **	0/0	d, es,*
day 8	0/0	slight d, es	0/0	slight d, es	0/0	slight d, es, **	0/0	slight d, es, **	0/0	slight d, es, **
day 9	0/0	slight d, es	0/0	slight d, es, **	0/0	slight d, es, **	0/0	slight d, es, **	0/0	slight d, es, **
day 10	0/0	slight d, es	0/0	slight d, es, s	0/0	slight d, es, **	0/0	slight d, es, **	0/0	slight d, es, **
day 11	0/0	moderate d, s#	0/0	slight d	0/0	slight d, es, **, s	0/0	slight d, es, s, **	0/0	slight d, es, **, s
day 12	0/0	slight d, es, s#	0/0	slight d	0/0	slight d, es, **, s	0/0	slight es, s, **	0/0	d, es, **, s
day 13	0/0	slight d, es, s#	0/0	slight d	0/0	slight d, es, **, s	0/0	slight es, s, **	0/0	d, es
day 14	0/0	es	0/0	slight d	0/0	es	0/0	es	0/0	d, es
day 15	0/0	es	0/0	nf	0/0	es	0/0	es	0/0	nf
Comments: E = erythema; O = oedema; 1, 2, 3, 4 = scoring system laid down in OECD Guideline 404 (Table 2) d = desquamation; es = eschar; s = scratches; s#: punctiform scratches, nf = no findings * orange discoloration at the application site, caused by the test item ** small wound

Interpretation of results:

not classified

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present study, single dermal application of phenothiazine to rats at a dose of 2000mg/kg body weight was associated with no signs of toxicity or mortality, but with signs of irritation. The dermal LD50 was determined to be greater than 2000mg/kg body weight. Phenothiazine does not require classification for acute percutaneous toxicity according to the provisions laid down in Regulation (EC) No 1272/2008.

Executive summary:

- Phenothiazine was tested for acute dermal Toxicity according to OECD guideline 402 and according to Regulation (EC) No 440/2008, Method B.3.

- Study was conducted as limit test (2000mg/kg body weight)

- 5 male and 5 female Wistar rats (Crl: WI(HAN)) were used in this study

- Corn oil was used as vehicle

- Rats were dermal exposed to phenothiazine for 24hrs

- Signs of toxicity related to dose level used, time of onset and duration were not observed (no treatment-related effects)

- No effects on organs (related to dose level) were observed

- LD50 is greater than 2000mg/kg body weight

- Signs of irritation were found in some animals. Not all signs were reversible within the observation period.

- Phenothiazine does not require classification for acute percutaneous toxicity according to the provisions laid down in Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Additional information

Two acute toxicity studies via oral route of exposure were considered for the present registration dossier (see IUCLID 5). One of these studies was conducted according to GLP and to previous OECD guidelines, while the other study was conducted as a standard acute method, but before implementation of GLP and development of OECD guidelines. While the OECD study resulted in a LD50 of more than 2000 mg/kg body weight, the pre-OECD study resulted in a LD50 of 1370 mg/kg body weight. In the OECD study there is no information on the purity of the test item provided, while a 99.8 % quality was tested as part of the pre-OECD study, which is similar to the > 99.5 % quality being registered as part of this dossier. Since there is no reason to doubt the results and the relevance of the pre-OECD study it was decided to use this study as the key study for this endpoint, hence accepting the resulting classification.

Regarding dermal toxicity one newly conducted limit test according to OECD 402 and GLP was available. In this study no signs of systemic toxicity were observed. However, skin irritation was observed during this study. Compared to the negative outcome of the studies on skin irritation this may be due to the choice of the vehicle. As part of this study DMSO was used, which dissolves phenothiazine, while water is used as vehicle for skin irritation studies. Under the latter conditions, phenothiazine is not dissolved. Use of DMSO in this study makes sense, since systemic toxicity after dermal application should be investigated and not primary irritant effects.

To fulfil registration requirements according to REACH, Annexes VII - X, two acute studies assessing two routes of exposure are needed. Based on physico-chemical properties of phenothiazine and the particle size of the primary product, inhalation is a less likely route of exposure for phenothiazine prills.

Justification for classification or non-classification

According to the present acute toxicity studies via oral route of exposure, classification as R22: harmful if swallowed (according to 67/548/EEC) or H302: Harmful if swallowed (according to 1272/2008/EC) is appropriate.

Based on the acute toxicity study via dermal route of exposure no classification is necessary either according to 67/548/EEC or 1272/2008/EC.

No classification is proposed for acute toxicity via inhalation, because physico-chemical data including particle size makes inhalation a less likely route of exposure.