Nitrile hydratase recombinant from E.coli

Endpoint summary

Description of key information

Skin sensitisation - in vitro (waiver)

According to Annex VII, column 2, standard information requirement 8.3.1, an in vitro skin sensitisation study does not need to be conducted if an in vivo study according to point 8.3.2 is available. Column 2, point 8.3.2 states that in vivo skin sensitisation studies that were carried out or initiated before 10 May 2017, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address this standard information requirement. Additionally, the murine local lymph node assay (LLNA) is the first- choice method for in vivo testing. Only in exceptional circumstances should another test be used. Justification for the use of another in vivo test shall be provided.

 

In the present case, a suitable guinea pig maximisation test (Skin sensitisation - Maximisation Test. Takahashi (2001)) is available, which meets the requirements set out in Article 13(3), first subparagraph, and Article 13(4). Additionally, the original Test Guideline (TG) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; TG 429) was adopted in 2002. Therefore, the LLNA method was not available by the time this guinea pig maximisation test (Skin sensitisation - Maximisation Test. Takahashi (2001)) was conducted (study period: 1 December 2000 to 30 December 2000).

 

Skin sensitisation - Maximisation Test. Takahashi (2001)

Under the conditions of this study, the test material was found not to be sensitising to the skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1 December 2000 to 30 December 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The original Test Guideline (TG) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; TG 429) was adopted in 2002. Therefore, the LLNA method was not available by the time this study was conducted (study period: 1 December 2000 to 30 December 2000).

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: 4 weeks old
- Weight at study initiation: 299 to 356 g (preliminary study), 289 to 328 g (main study)
- Housing: Animals were housed 1 or 2 per aluminum cage (38 cm [D] x 30 cm [W] x 18 cm [H]) with bedding in the SPF animal facility guinea pig study room.
- Diet: Approximately 25 g of irradiated sterilised solid food for guinea pigs was provided once daily to each animal. Each lot of food was analysed, and it was confirmed that there were no problems.
- Water: Water conforming to the quality standards in the Japanese Water Supply Act was available ad libitum in 200 mL water bottles. Water was analysed regularly and it was confirmed that there were no problems.
- Acclimation period: 10 days (preliminary study), 7 days (main study)
- Animal health: During the 7-day quarantine and acclimation period, the animals were observed for clinical signs once daily and weighed once on the first and
final days. Twenty males that showed no abnormalities were used (main study).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Temperature in the room was set at 22 ± 2°C (actual range: 20.5 to 23.8°C, acceptable range: 18 to 26°C)
- Humidity (%): Humidity set at 50 ± 10% (actual range: 39 to 64%, acceptable range: 30 to 70%),
- Air changes (per hr): 15 times/hour
- Photoperiod (hrs dark / hrs light): 12 hours of artificial light per day (lights on from 6:00 am to 6:00 pm)
- Additional information: The study room was cleaned daily, and the cages, bedding, water bottles, and food containers were exchanged twice weekly for autoclaved (121°C, 30 minutes) replacements.

Route:

intradermal

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 vol%. 0.1 mL/site

Adequacy of induction:

highest technically applicable concentration used

Route:

epicutaneous, semiocclusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100 vol%, filter papers (2 x 4 cm) loaded with 0.2 mL of preparation were applied

Day(s)/duration:

Topical induction begins 7 days after intradermal induction. Exposure duration: 48 hours

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, semiocclusive

Vehicle:

water

Remarks:

water for injection

Concentration / amount:

100 (undiluted solution), 10, 1, 0.1, and 0.01 vol% concentrations. 1.5 x 1.5 cm filter papers loaded with 0.1 mL/site of 100, 10, 1, 0.1, and 0.01 w/v% test article preparations were applied

Day(s)/duration:

Challenge exposure occurred 12 days after topical induction. Exposure duration: 24 hours

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

The study design consisted of 1 test article group, 1 negative control group, and 1 positive control group (total: 3 groups). 10 animals for the test article group and 5 animals each for the negative and positive control groups were selected.

In the test article and negative control article groups, 100, 10, 1, 0.1, and 0.01 w/v% test article preparations were applied to sites D to H (see illustration included in 'Overall remarks, attachments' section) and a 1.5 x 1.5 cm filter paper loaded with water for injection was applied to site I. Therefore, each animal in the test article (10 animals) and negative control article (5 animals) groups was exposed to 5 different concentrations as well as water for injection only during challenge.

Details on study design:

PRELIMINARY STUDY:
- Study Design and Identification of Animals:
The study consisted of 2 groups. On the final day of the quarantine and acclimation period (November 30, 2000), 6 animals were selected randomly for each group. The animals were identified by marking with dye during examination, and cages were identified by color-coded cage cards (indicating the study number, group number, and animal number). The study design is shown in Table 'Preliminary Study Design'.
- Study Method - Intradermal Dosing Group:
The back of each animal was shaved, and 0.1 mL each of the 100, 75, 50, 25, 10, and 5 vol% test article preparations was administered once intradermally with a disposable syringe and needle to the back of 2 guinea pigs (single dose).
- Study Method - Percutaneous Dosing Group:
The back of each animal was shaved, and filter papers (1.5 x 1.5 cm) which had been loaded with 0.2 mL of 100, 75, 50, 25, 10, and 5 vol% test article preparation had been applied were applied to 2 guinea pigs. The filter papers were then covered with Parafilm, secured with gauze and adhesive tape, and covered with a bandage once (single dose) for 24 hours.
- Clinical Signs Observation:
Clinical signs and mortality were observed once daily from the dosing day until the final day of observation in all animals. Clinical signs were observed once before and once after dosing on the dosing day.
- Evaluation:
Skin reaction was evaluated in accordance with the Draize method criteria.(1) The intradermal induction concentration for the main study was set at the highest concentration at which no eschar was observed. The lowest concentration at which erythema was observed was taken as the topical induction concentration. The highest concentration at which no erythema was observed was taken as the challenge concentration. See Table 'Criteria for the Evaluation of Skin Reaction' for further details.

MAIN STUDY
- Study Design and Identification of Animals:
The study design consisted of 1 test article group, 1 negative control group, and 1 positive control group (total: 3 groups). On the day before intradermal induction (December 4, 2000), animals were randomly assigned by stratified randomization according to body weight; 10 animals for the test article group and 5 animals each for the negative and positive control groups were selected. The animals were identified by marking with dye during the quarantine and acclimation period and by ear punching after grouping. Cages were identified by color-coded cage cards (indicating the study number, group number, and animal number). See Table 'Main Study Design' for further details.
- Justification for Selection of the Dose Concentration:
As a result of the preliminary study, eschar was not observed in the intradermal dosing group at any concentration. Therefore, the intradermal induction concentration in the main study was set at 100 vol%. In the percutaneous dosing group, no erythema was observed at any concentration. Therefore, the topical induction concentration was set at 100 vol%, and the challenge concentration was set at 100 vol% (undiluted solution) as the highest concentration, at 10, 1, 0.1 and 0.01 vol% in a common ratio of 10.
- Clinical Signs Observation:
Clinical signs and mortality were observed once daily from the first day of induction until the final day of observation in all animals. Clinical signs were observed once before and once after dosing on the days of intradermal induction, topical induction, and challenge dosing.

A. INDUCTION EXPOSURE
- Intradermal Induction:
The induction sites on the dorsal scapular region of the guinea pig were shaved to a size of 4 x 6 cm with electric hair clippers (0.5 mm blade) on the day before dosing, and the 3 preparations stated below were injected once (single dose) intradermally with a disposable syringe and needle at a volume of 0.1 mL/site to the left and right sides at paired sites A, B, and C.
A: 1:1 W/O emulsion of water for injection and FCA
B: Test article preparation (Group 1: Vehicle-1, Group 2: Test material preparation, Group 3: 0.1 w/v% DNCB solution)
C: Test article or control article-FCA mixture (Group 1: W/O emulsion of vehicle-1 and FCA, Group 2: W/O emulsion of test material preparation and FCA, Group 3: W/O emulsion of 0.2 w/v% DNCB solution and FCA)

- Topical Induction:
The same area as for intradermal induction was shaved 7 days after intradermal induction. 10% Sodium lauryl sulfate ointment (0.5 g) as an inflammatory agent was administered by direct application at the intradermal induction sites. The inflammatory agent was removed after 24 hours, and filter papers (2 x 4 cm) loaded with 0.2 mL of preparation were applied. The filter papers were then covered with Parafilm, secured with gauze and adhesive tape, and covered with a bandage for 48 hours. The frequency and period was counted as one (single) dosing.

B. CHALLENGE EXPOSURE
For the challenge sites, areas approximately 8 cm wide on the left and right abdomen, as shown in the illustration included in 'Overall remarks, attachments' section, were shaved with electric clippers 11 days after topical induction. On the following day (12 days after topical induction), 1.5 x 1.5 cm filter papers loaded with 0.1 mL/site of 100, 10, 1, 0.1, and 0.01 w/v% test article preparations were applied to sites D to H and a 1.5 x 1.5 cm filter paper loaded with water for injection was applied to site I in the test article and negative control article groups. In the positive control article group, a 1.5 x 1.5 cm filter paper loaded with 0.1 mL/site of 0.1 w/v% DNCB solution was applied to site E. The filter papers were then covered with Parafilm, secured with gauze and adhesive tape, and covered with a bandage for 24 hours. The frequency and period was counted as one (single) dosing.
- Evaluation (hr after challenge):
Skin reaction at 24 and 48 hours after challenge was determined. Skin reaction was evaluated in accordance with the Draize method criteria.(1) See Table 'Criteria for the Evaluation of Skin Reaction' for further details. Photographs were taken of one representative animal per group.
- Body Weight:
Animals were weighed on the evaluation day of 48 hours after challenge.

JUSTIFICATION FOR SELECTION OF THE DOSING ROUTE, VOLUME, METHOD, FREQUENCY AND PERIOD
Dosing route, volume, method, frequency, and period for intradermal induction, topical induction, and challenge were all in accordance with the OECD Guidelines for Testing of Chemicals 406 (Skin Sensitisation, adopted: 17th July 1992).

TREATMENT OF ANIMALS
Surplus animals after grouping and study animals after completion of macroscopic observations were euthanized by an intraperitoneal injection of 4 mL/body of sodium pentobarbital (Tokyo Kasei Kogyo Co., Ltd.) solution (64.8 mg/mL).

EVALUATION OF RESULTS AND STATISTICAL ANALYSIS
Statistical analysis was not performed.

References:
(1) Draize JH, Woodard G, and Calvery H. 0.: Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J. Pharmacol. Exp. Ther.;82:377-390, 1944

Positive control substance(s):

yes

Remarks:

2,4-Dinitrochlorobenzene (DNCB)

Positive control results:

In the positive control group, erythema/eschar scored as 3 or 4 and edema scored as 4 was observed 24 or 48 hours after challenge. The mean reaction score 24 hours after challenge was 7.2. The mean reaction score 48 hours after challenge was 7.6.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

positive indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1%

No. with + reactions:

5

Total no. in group:

5

Clinical observations:

No abnormalities were noted in clinical signs or body weight change in any animal in any group during the observation period.

Remarks on result:

positive indication of skin sensitisation

Mean Reaction Scores

Group	Number of animals	Treatment			Score*
		Induction		Challenge	24 hours**			48 hours**
		lntradermal injection	Topical application		Erythema	Edema	Total	Erythema	Edema	Total
1	5	Water for injection	Water for injection	Water for injection	0.0	0.0	0.0	0.0	0.0	0.0
				0.01 % Test material	0.0	0.0	0.0	0.0	0.0	0.0
				0.1% Test material	0.0	0.0	0.0	0.0	0.0	0.0
				1 % Test material	0.0	0.0	0.0	0.0	0.0	0.0
				10% Test material	0.0	0.0	0.0	0.0	0.0	0.0
				100% Test material	0.0	0.0	0.0	0.0	0.0	0.0
2	10	Test material	Test material	Water for injection	0.0	0.0	0.0	0.0	0.0	0.0
				0.01 % Test material	0.0	0.0	0.0	0.0	0.0	0.0
				0.1% Test material	0.0	0.0	0.0	0.0	0.0	0.0
				1 % Test material	0.0	0.0	0.0	0.0	0.0	0.0
				10% Test material	0.0	0.0	0.0	0.0	0.0	0.0
				100% Test material	0.0	0.0	0.0	0.0	0.0	0.0
3	5	0.1% DNCB	0.1% DNCB	0.1% DNCB	3.2	4.0	7.2	3.6	4.0	7.6

* Total irritation score/number of animals

** Time (hour) after the removal of challenge

Erythema: Erythema and eschar

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of this study, the test material was found not to be sensitising to the skin.

Executive summary:

The skin sensitisation potential of the test material was investigated in guinea pigs using the maximisation method according to OECD guideline 406 and in compliance with GLP. A guinea maximisation study was performed, as opposed to a LLNA study, because the LLNA method was not available by the time this study was conducted (study period: 1 December 2000 to 30 December 2000).

 

In the test article group, 100, 10, 1, 0.1, and 0.01 w/v% of the test material and water for injection were applied to guinea pigs sensitised intradermally and by topical application for 24 hours. The challenge sites were observed 24 and 48 hours after challenge. In the negative control group, water for injection was injected intradermally and applied topically, and the animals were challenged in the same manner as those in the test material group. In the positive control group, 0.1 w/v% DNCB solution was injected intradermally and applied topically, and the animals were challenged with 0.1 w/v% DNCB solution.

 

In the test material group, no abnormalities were observed at any challenge site in all animals at any observation point. In the negative control group, no abnormalities were observed at any challenge site at any observation point. In the positive control group, clear positive reactions (erythema/eschar and edema) were observed at all observation points.

 

Under the conditions of this study, the test material was found not to be sensitising to the skin.

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
According to Annex VII, column 2, standard information requirement 8.3.1, an in vitro skin sensitisation study does not need to be conducted if an in vivo study according to point 8.3.2 is available. Column 2, point 8.3.2 states that in vivo skin sensitisation studies that were carried out or initiated before 10 May 2017, and that meet the requirements set out in Article 13(3), first subparagraph, and Article 13(4) shall be considered appropriate to address this standard information requirement. Additionally, the murine local lymph node assay (LLNA) is the first- choice method for in vivo testing. Only in exceptional circumstances should another test be used. Justification for the use of another in vivo test shall be provided.

In the present case, a suitable guinea pig maximisation test (Skin sensitisation - Maximisation Test. Takahashi (2001)) is available, which meets the requirements set out in Article 13(3), first subparagraph, and Article 13(4). Additionally, the original Test Guideline (TG) for the determination of skin sensitisation in the mouse, the Local Lymph Node Assay (LLNA; TG 429) was adopted in 2002. Therefore, the LLNA method was not available by the time this guinea pig maximisation test (Skin sensitisation - Maximisation Test. Takahashi (2001)) was conducted (study period: 1 December 2000 to 30 December 2000).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.

 

In accordance with Annex VI, regulation (EC) No 1272/2008, the substance does require classification with respect to respiratory sensitisation - Category 1: H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled.