Nitrile hydratase recombinant from E.coli

Endpoint summary

Description of key information

Skin irritation in vivo - key study (Takahashi, 2003)

Under the conditions of this study, the test material causes no skin irritation.

 

 

Skin irritation in vitro (waiver)

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

 

 

Eye irritation (waiver)

In accordance with REACH Annex XI, section 2, an eye irritation study is not required as testing is technically not possible as a consequence of the properties of the substance and it is not considered either necessary or appropriate to conduct either an in vitro, or an in vivo, eye irritation study.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

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

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
An in vitro skin irritation study does not need to be conducted because adequate data from an in vivo skin irritation study are available.

Reference 2

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2003 to 29 September 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

2002

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

other: Japanese white

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 17 weeks
- Weight at study initiation: 3.06-3.20 kg
- Housing: Rabbits were housed in stainless steel or aluminium cages. (63 cm (D) x 50 cm (W) x 36 cm (H)). There were one rabbit per cage.
- Diet: approx. 120 g of solid fodder was given once a day
- Water: ad libitum
- Acclimation period: 8 Days

ENVIRONMENTAL CONDITIONS
- Temperature: 21.5 – 22.6°C
- Humidity: 43 – 66%
- Air changes: 15 times/hour
- Photoperiod: 12 hours/day artificial lighting (06:00 – 18:00 lamp-lighting)

Type of coverage:

occlusive

Preparation of test site:

clipped

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent no treatment

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 0.5 mL per site

Duration of treatment / exposure:

3 minutes, 1, 4 hours

Observation period:

1, 24, 48 and 72 hours

Number of animals:

3 animals

Details on study design:

TEST SITE
- Area of exposure: Test material was coated uniformly on 2.5 x 2.5 cm lint cloth
- Type of wrap if used: Lint cloth was covered with paraffin film and fixed with gauze and an adhesive plaster, covered with bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: Applied area was wiped with gauze dipped in warm water
- Time after start of exposure: 3 minutes initially on animal No.1. Since no formation of crust, the test material was applied for 4 hours to the remaining sections on animals No.1 to 3.

OBSERVATION TIME POINTS
- Pasted sections of all the three animals with the test material were judged and evaluated after 1, 24, 48 and 72 hours from the removal of the pastings.

SCORING SYSTEM:
- Method of calculation: Draize method

Irritation parameter:

erythema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

erythema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritation parameter:

edema score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Irritant / corrosive response data:

The individual scores for erythema/eschar and edema are given in Table 'Average irritation score on skin - part 1' and Table 'Average irritation score on skin - part 2'. The skin to which the test material was pasted for 3 minutes, 1 hour and 4 hours showed no abnormality in all of the animals during any of the observation periods.

Average irritation score on skin - part 1

Animal no.	Treatment	Item	Administration time (minute)
			3	60
1	Test Material	Erythema and eschar	0	0
		Edema	0	0
		Total	0	0

 

Average irritation score on skin - part 2

Animal No.	Treatment	Patched sites	Item	Time (hour) after removed of patch				P.I.I.	Irritation grade
				1	24	48	78
1	Test Material	left	Erythema and eschar	0	0	0	0	0.0	-
			Edema	0	0	0	0
			Total	0	0	0	0
2	Test Material	left	Erythema and eschar	0	0	0	0
			Edema	0	0	0	0
			Total	0	0	0	0
3	Test Material	left	Erythema and eschar	0	0	0	0
			Edema	0	0	0	0
			Total	0	0	0	0
Mean				0.0	0.0	0.0	0.0
1	Non-treatment	right	Erythema and eschar	0	0	0	0	0.0	-
			Edema	0	0	0	0
			Total	0	0	0	0
2	Non-treatment	right	Erythema and eschar	0	0	0	0
			Edema	0	0	0	0
			Total	0	0	0	0
3	Non-treatment	right	Erythema and eschar	0	0	0	0
			Edema	0	0	0	0
			Total	0	0	0	0
Mean				0.0	0.0	0.0	0.0

Interpretation of results:

other: Not classified according to EU criteria

Conclusions:

Under the conditions of this study, the test material caused no skin irritation.

Executive summary:

The potential of the test material to cause skin irritation was investigated in accordance with the standardised guideline OECD 404, under GLP conditions.

The test material was pasted under sealed condition to the skin of Japanese white rabbits for 3 minutes, 1 hour and 4 hours, and its skin irritation was investigated. All of the animals showed no abnormality on their skin to which the test material was pasted under sealed condition for 3 minutes, 1 hour and 4 hours during each of the observation periods. The primary irritation index P.I.I. as calculated from the average of the marks for 24, 48 and 72 hours after the removal of KNH-4 was 0.0. During the observation periods, none of the animals showed any abnormality in their general conditions.

Under the conditions of this study, the test material caused no skin irritation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with REACH Annex XI, section 2, an eye irritation study is not required as testing is technically not possible as a consequence of the properties of the substance. The substance, nitrile hydratase, is a substance that is handled in a state where a mixture of the enzyme and the dead bacteria that produced it is dispersed in water. A fundamental requirement for testing the physicochemical properties of a substance, is that the substance is tested as such, excluding all solvents. However, since the higher order structure of the constituent molecules of the proposed substance (nitrile hydratase) is damaged by the removal of water, it cannot be said that the proposed substance would be correctly evaluated by performing this test after the removal of water. It should therefore be avoided.

In order to avoid deterioration it would be possible to conduct the test by leaving the substance as a suspension (i.e. dispersed in water). However, to do so would render the test results meaningless as the physical properties of water would greatly influence the results of such a test. As such, it would not be possible to consider the results of any such test as indicative of the physicochemical properties of the substance as such.

Additionally, there are substantial in vivo animal data on all industrially applied enzyme classes, as well as in vitro data performed before formal validation, for both skin and eye irritancy. These data document that enzymes are not irritating, with the exception of proteases. Non-proteases lack the potential to be skin and eye irritants but proteases show a range of effects from no irritation to moderate irritation. It is, however, only the most concentrated and reactive samples which show an effect which is transient in nature and commonly is classified as mild irritation (1; 2; in-house industrial data). This is also the outcome of clinical investigations (3; 4; 5), confirming that enzymes at the product use concentration, even with exaggerated exposures, do not give rise to any occupational or consumer risk of skin or eye irritation. Given the above, 'in vitro’ eye irritation testing is not considered to add any scientific value nor lead to a change in the classification adopted from existing data (in-house industrial data).

References:
1. AMFEP. Amfep paper on safety evaluation of technical enzyme products with regards to the REACH legislation. 2009. AMFEP.
2. HERA Human and environmental risk assessment on ingredients of household cleaning products - alpha-amylases, cellulases and lipases. 2005.
3. Basketter, D.A., English, J.S., Wakelin, S.H., and White, I.R. (2008) Enzymes, detergents and skin: facts and fantasies. British journal of dermatology 158, 1177-1181
4. Griffith, J.F., Weaver, J.E., Whitehouse, H.S., Poole, R.L., Newmann, E.A. and Nixon, G.A. (1969) SAFETY EVALUATION OF ENZYME DETERGENTS ORAL AND CUTANEOUS TOXICITY IRRITANCY AND SKIN SENSITIZATION STUDIES. Food and Cosmetics Toxicology 7, 581-593
5. White, I.R., Lewis, J. and El Alami, A. (1985) Possible adverse reactions to an enzyme-containing washing powder. Contact Dermatitis 13, 175-179

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation in vivo - key study (Takahashi, 2003)

The potential of the test material to cause skin irritation was investigated in accordance with the standardised guideline OECD 404, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

 

The test material was pasted under sealed condition to the skin of Japanese white rabbits for 3 minutes, 1 hour and 4 hours, and its skin irritation was investigated. All of the animals showed no abnormality on their skin to which the test material was pasted under sealed condition for 3 minutes, 1 hour and 4 hours during each of the observation periods. The primary irritation index P.I.I. as calculated from the average of the marks for 24, 48 and 72 hours after the removal of KNH-4 was 0.0. During the observation periods, none of the animals showed any abnormality in their general conditions.

 

Under the conditions of this study, the test material caused no skin irritation.

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 or eye irritation.