Registration Dossier

Administrative data

Workers - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
97.95 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
18
Modified dose descriptor starting point:
NOAEC
Value:
1 763 mg/m³
Explanation for the modification of the dose descriptor starting point:
Correction of the starting point according ECHA Guidance Chapter R.8: There are no quantitative data on absorption rates for oral or for inhalation route available. Based on the information that the substance is good soluble in water it is assumed that absorption rate via inhalation route is in the same range as the absorption via oral route. Therefore there is no need to introduce further default value. Corrected inhalatory NOAEC = Oral NOAEL (1000 mg/kg) x 1/0.38 m³/kg x 6.7 m³/10m³ x 1 = 1763 mg/m³
AF for dose response relationship:
1
Justification:
There are no effects up to the limit dose
AF for differences in duration of exposure:
6
Justification:
Sub-acute to chronic exposure
AF for interspecies differences (allometric scaling):
1
Justification:
Allometric scaling: rat versus human the AF of 4 is already included in the route to route extrapolation
AF for other interspecies differences:
1
Justification:
The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified
AF for intraspecies differences:
3
Justification:
Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the sub-acute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.
AF for the quality of the whole database:
1
Justification:
There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a guideline sub-acute toxicity study indicating that Nigrosin has a low toxicological potential and the starting point for the DNEL derivation is conservative
AF for remaining uncertainties:
1
Justification:
In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
195.9 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
DNEL extrapolated from long term DNEL
Explanation for the modification of the dose descriptor starting point:
There is no information available to evaluate acute inhalation toxicity. Therefore, short term inhalation cannot be assessed nor be allocated to a hazard band. In addition, as there is no inhalation study available using repeated exposure the long term DNEL for inhalation route was derived from the available subacute oral study in animals dosed up to and including 1000 mg/kg bw/day and which was tolerated without impairment. According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (inhalation, long term) to DNEL (inhalation ,short term) if no data for acute inhalation toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied.

Local effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

Workers - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
13.9 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
72
Modified dose descriptor starting point:
NOAEL
Value:
1 000 mg/kg bw/day
Explanation for the modification of the dose descriptor starting point:
According to ECHA guidance document No R8 Version 2, December 2010, no additional default factor has to be introduced when performing oral to dermal extrapolation because it can be assumed that dermal absorption will not be higher that oral absorption: NOAEL (subacute, oral) = NOAEL (subacute, dermal) = 1000 mg/kg bw/day
AF for dose response relationship:
1
Justification:
There are no effects up to the limit dose
AF for differences in duration of exposure:
6
Justification:
Subacute to chronic exposure
AF for interspecies differences (allometric scaling):
4
Justification:
Allometric scaling rat versus human
AF for other interspecies differences:
1
Justification:
The rats tolerated the dosing up to and including the limit dose 1000 mg/kg bw is a conservative starting point for DNEL calculation and the real NOAEL in rats is much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified
AF for intraspecies differences:
3
Justification:
Based on the consideration by ECETOC Report TR110, 2010: furthermore, there are no toxic effects up to the limit dose. . In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.
AF for the quality of the whole database:
1
Justification:
There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP
AF for remaining uncertainties:
1
Justification:
In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
27.8 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
DNEL extrapolated from long term DNEL
Explanation for the modification of the dose descriptor starting point:
There are acute toxicity studies available using the dermal exposure route. These studies comprise acute toxicity, skin irritation and skin sensitization. In none of the three study types mortality or signs of intoxication were observed. The LD50 (dermal) is greater 2000 mg/kg bw and the test substance was not irritating to the skin nor caused skin sensitization. Thus, Nigrosin WLF cannot be allocated to a hazard band. As the NOAEL for acute dermal toxicity is not determined exactely it is proposed not to derive a DNEL for short term exposure by taking the derived DNEL (systemic, long term, dermal route) as starting point.According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (dermal, long term) to DNEL (dermal, short term) if no exact data for acute dermal toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied. Thus, the DNEL (systemic, short term, dermal route) is about 27.8 mg/kg bw/day

Local effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
skin irritation/corrosion

Workers - Hazard for the eyes

Local effects

Hazard assessment conclusion:
no hazard identified

Additional information - workers

I.Introduction

Nigrosin WLF is not classified/labeled legally and there is no German MAK or European SCOEL available which could be taken as starting point for the delineation of DNELs:

II.Derivation of DNELs systemic

Basis for delineation of the DNELs (systemic)

Nigrosin WLF dissolved in tap water, was administered orally by gavage in doses of 0, 100, 300 and 1000 mg/kg bw/day to 5 male and 5 female Wistar rats per dose group for a period of 4 weeks according to OECD TG 407 and GLP. Survival, body weight development as well as food and water intake in treated groups were not affected by treatment, Neither hematology nor clinical chemistry gave evidence for treatment related effects up to 1000 mg/kg bw/day. Test item related macroscopic and microscopic changes at terminal sacrifice were slight grey or black discolorations in a dose-related manner in all treated groups, predominantly in the lymph nodes, and less frequently in the intestine and/or Peyer's patch. Corresponding to these findings at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups. All these discolorations were considered to be due to the staining property of the test item. In the view of the very limited degree of changes and as any indication of structural change or functional impairment of the organs concerned, was lacking, they were considered to be non-adverse.

Therefore, under the conditions described, the NOAEL for administration of Nigrosin WLF to male and female rats was 1000 mg/kg bw/day.

1.)DNEL Long-term, inhalation route – systemic effects (worker) using extrapolation factors:

NOAEL (rat) from a subacute oral toxicity study:1000 mg/kg bw/day

Correction of the starting point according ECHA Guidance Chapter R.8: There are no quantitative data on absorption rates for oral or for inhalation route available. Based on the information that the substance is good soluble in water it is assumed that absorption rate via inhalation route is in the same range as the absorption via oral route. Therefore there is no need to introduce further default value.

Corrected inhalatory NOAEC = Oral NOAEL (1000 mg/kg) x 1/0.38 m³/kg x 6.7 m³/10m³ x 1 => NOAEC worker = 1763.15 mg/m³

Factors to be applied and Justification

----AF for dose response relationship:      1                                   

There are no effects up to the limit dose

----AF for differences in duration of exposure       6

Sub-acute to chronic exposure

---AF for interspecies differences 1

Allometric scaling: rat versus human the AF of 4 is already included in the route to route extrapolation

----AF for intraspecies differences: worker 3

Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the sub-acute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.”. Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.

----AF for other interspecies differences 1

The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified.

----AF for quality of the whole database 1

There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a guideline sub-acute toxicity study indicating that Nigrosin has a low toxicological potential and the starting point for the DNEL derivation is conservative.

----AF for remaining differences 1

In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)

Overall factor : 18

Worker DNEL (long term, systemic for inhalation route) 97.95 mg/m³

2.)DNEL (Short-term, inhalation route – systemic effects)

There is no information available to evaluate acute inhalation toxicity. Therefore, short term inhalation cannot be assessed nor be allocated to a hazard band. In addition, as there is no inhalation study available using repeated exposure the long term DNEL for inhalation route was derived from the available subacute oral study in animals dosed up to and including 1000 mg/kg bw/day and which was tolerated without impairment. According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (inhalation, long term) to DNEL (inhalation ,short term) if no data for acute inhalation toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied.

Thus, the DNEL (systemic, short term, inhalation route) is about 195.90 mg/m³

3) DNEL (Long-term, dermal route-systemic effects)

According to ECHA guidance document No R8 Version 2, December 2010, no additional default factor has to be introduced when performing oral to dermal extrapolation because it can be assumed that dermal absorption will not be higher that oral absorption: NOAEL (subacute, oral) = NOAEL (subacute, dermal) = 1000 mg/kg bw/day

Factors to be applied Justification

----AF for dose response relationship 1

There are no effects up to the limit dose

----AF for differences in duration of exposure 6

Subacute to chronic exposure:

----AF for interspecies differences 4

Allometric scaling: rat versus human

----AF for intraspecies differences: worker 3

Based on the consideration by ECETOC Report TR110, 2010:

Furthermore, there are no toxic effects up to the limit dose. . In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.

----AF for other interspecies differences 1

The rats tolerated the dosing up to and including the limit dose 1000 mg/kg bw is a conservative starting point for DNEL calculation and the real NOAEL in rats is much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified

----AF for quality of the whole database 1

There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP

----AF for remaining differences 1

In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)

Overall factor 72

Worker DNEL long-term, systemic for dermal route: 13.89 mg/kg bw/day

4.)DNEL (Short-term dermal route – systemic effects)

There are acute toxicity studies available using the dermal exposure route. These studies comprise acute toxicity, skin irritation and skin sensitization. In none of the three study types mortality or signs of intoxication were observed. The LD50 (dermal) is greater 2000 mg/kg bw and the test substance was not irritating to the skin nor caused skin sensitization. Thus, Nigrosin WLF cannot be allocated to a hazard band. As the NOAEL for acute dermal toxicity is not determined exactely it is proposed not to derive a DNEL for short term exposure by taking the derived DNEL (systemic, long term, dermal route) as starting point. According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (dermal, long term) to DNEL (dermal, short term) if no exact data for acute dermal toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied. Thus, the DNEL (systemic, short term, dermal route) is about 27.8 mg/kg bw/day

5.)DNEL (Reproductive toxicity - Fertility assessment)

There is no specific investigation available to evaluate the effects on fertility. In the available subacute toxicity study with oral application of doses up to and including 1000 mg/kg bw/day over a period of 28 days no adverse effects on reproductive organs are reported. Therefore, it is assumed fertility is not affected by treatment with Nigrosin WLF and the NOAEL for fertility assessment complies with the NOAEL for general toxicity.

Based on this consideration no additional DNEL has to be derived.

6.) DNEL (Reproductive Toxicity - Assessment of development)

In a developmental toxicity study according OECD TG 414 the reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No dead fetuses and no test item-related malformations, variations or retardations were noted. Under the conditions of the study, Nigrosin WLF did not show any teratogenic potential.

The no-observed-adverse effect level (NOAEL) for the fetal organism was above 1000 mg Nigrosin WLF/kg bw/day.

III. Delineation of DNEL (local)

Basis for delineation of the DNELs (local)

According to ETAD Sub-komitee für Toxikologie: Methoden für toxikolog. Untersuchungen von Farbstoffen und Hilfsmitteln Nigrosin WLF was tested for skin irritation and for irritation of the mucous membranes of the eyes. No irritation effects were observed neither to the skin of rabbits nor in the mucous membranes of the eyes.

Furthermore, the modified Local Lymph Node Assay (IMDS) was performed according to OECD Guidelines No. 429 and No. 406. The results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % Nigrosin WLF in this test system. No effects are observed.

Based on these results, no DNELs (local) need to be derived because no hazard was identified.

IV Conclusion (systemic and local effects):

The systemic DNELs for long term exposure include the short term hazard. Therefore, the relevant DNELs are

Worker DNEL (systemic, inhalation route): 97.95 mg/m³

Worker DNEL (systemic, dermal route): 13.89 mg/kg bw/day

With respect to local exposure no DNELs are needed because no hazard was identified.

General Population - Hazard via inhalation route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
29 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
30
Modified dose descriptor starting point:
NOAEC
Value:
870 mg/m³
Explanation for the modification of the dose descriptor starting point:
There are no quantitative data on absorption rates for oral or for inhalation route available. Based on the information that the substance is good soluble in water it is assumed that absorption rate via inhalation route is in the same range as the absorption via oral route. Therefore there is no need to introduce further default value Corrected inhalatory NOAEC = Oral NOAEL (1000 mg/kg) x 1/1.15 m³/kg x 1 => NOAEC general public = 870 mg/m³
AF for dose response relationship:
1
Justification:
there are no effects up to the limit dose
AF for differences in duration of exposure:
6
Justification:
Sub acute to chronic exposure
AF for interspecies differences (allometric scaling):
1
Justification:
Allometric scaling: rat versus human: the AF of 4 is already included in the route to route extrapolation
AF for other interspecies differences:
1
Justification:
The rats tolerated the dosing up to and including the limit dose 1000 mg/kg bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the fecesand no additional factorfor potential toxico-dynamicalterationin different animal species is justified
AF for intraspecies differences:
5
Justification:
Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the subacute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data
AF for the quality of the whole database:
1
Justification:
There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a sub-acute guideline study indicating that Nigrosin WLF has a low toxicological potential and and the starting point for the DNEL derivation is conservative
AF for remaining uncertainties:
1
Justification:
In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
58 mg/m³
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
DNEL extrapolated from long term DNEL

Local effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
DNEL related information

General Population - Hazard via dermal route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
8.33 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
120
Modified dose descriptor starting point:
NOAEL
Value:
1 000 mg/kg bw/day
Explanation for the modification of the dose descriptor starting point:
Considering dermal exposure route and taking into account ECHA guidance document No R8 Version 2, December 2010, no additional default factor has to be introduced when performing oral to dermal extrapolation because it can be assumed that dermal absorption will not be higher than oral absorption: NOAEL (subacute, oral) = NOAEL (subacute, dermal) = 1000 mg/kg bw/day
AF for dose response relationship:
1
Justification:
There are no effects up to the limit dose
AF for differences in duration of exposure:
6
Justification:
Subacute to chronic exposure
AF for interspecies differences (allometric scaling):
4
Justification:
Allometric Scaling rat versus human
AF for other interspecies differences:
1
Justification:
The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified
AF for intraspecies differences:
5
Justification:
Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the sub-acute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.
AF for the quality of the whole database:
1
Justification:
There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a guideline sub-acute toxicity study indicating that Nigrosin WLF has a low toxicological potential and the starting point for the DNEL derivation is conservative
AF for remaining uncertainties:
1
Justification:
In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
16.7 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
DNEL extrapolated from long term DNEL

Local effects

Long term exposure
Hazard assessment conclusion:
no hazard identified
Acute/short term exposure
Hazard assessment conclusion:
no hazard identified
Most sensitive endpoint:
skin irritation/corrosion
DNEL related information
DNEL derivation method:
ECHA REACH Guidance

General Population - Hazard via oral route

Systemic effects

Long term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
8.33 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
Overall assessment factor (AF):
120
Modified dose descriptor starting point:
NOAEL
Value:
1 000 mg/kg bw/day
Explanation for the modification of the dose descriptor starting point:
No route to route extrapllation performed
AF for dose response relationship:
1
Justification:
there are no effects up to the limit dose
AF for differences in duration of exposure:
6
Justification:
Subacute to chronic exposure
AF for interspecies differences (allometric scaling):
4
Justification:
Allometric scaling rat versus human
AF for other interspecies differences:
1
Justification:
The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified
AF for intraspecies differences:
5
Justification:
Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the sub-acute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces.As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.
AF for the quality of the whole database:
1
Justification:
There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a guideline sub-acute toxicity study indicating that Nigrosin WLF has a low toxicological potential and the starting point for the DNEL derivation is conservative.
AF for remaining uncertainties:
1
Justification:
In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010
Acute/short term exposure
Hazard assessment conclusion:
DNEL (Derived No Effect Level)
Value:
16.7 mg/kg bw/day
Most sensitive endpoint:
repeated dose toxicity
Route of original study:
Oral
DNEL related information
DNEL derivation method:
ECHA REACH Guidance
DNEL extrapolated from long term DNEL

General Population - Hazard for the eyes

Local effects

Hazard assessment conclusion:
no hazard identified

Additional information - General Population

I. Introduction

II. Derivation of DNELs systemic

Basis for delineation of the DNELs (systemic)

Nigrosin WLF dissolved in tap water, was administered orally by gavage in doses of 0, 100, 300 and 1000 mg/kg bw/day to 5 male and 5 female Wistar rats per dose group for a period of 4 weeks according to OECD TG 407 and GLP. Survival, body weight development as well as food and water intake in treated groups were not affected by treatment, Neither hematology nor clinical chemistry gave evidence for treatment related effects up to 1000 mg/kg bw/day. Test item related macroscopic and microscopic changes at terminal sacrifice were slight gray or black discolorations in a dose-related manner in all treated groups. All these discolorations were considered to be due to the staining property of the test item. In the view of the very limited degree of changes and as any indication of structural change or functional impairment of the organs concerned was lacking, they were considered to be non-adverse. Therefore, under the conditions described, the NOAEL for administration of Nigrosin WLF to male and female rats was 1000 mg/kg bw/day.

1.) DNEL Long-term, inhalation route – systemic effects (general public) using extrapolation factors:

NOAEL (rat) from a sub-acute oral toxicity study: 1000 mg/kg bw/day

Correction of the starting point according ECHA Guidance Chapter R.8:

There are no quantitative data on absorption rates for oral or for inhalation route available. Based on the information that the substance is good soluble in water it is assumed that absorption rate via inhalation route is in the same range as the absorption via oral route. Therefore there is no need to introduce further default value

Corrected inhalatory NOAEC = Oral NOAEL (1000 mg/kg) x 1/1.15 m³/kg x 1

=> NOAEC general public = 870 mg/m³

Factors to be applied and Justification

----AF for dose response relationship 1

There are no effects up to the limit dose

----AF for differences in duration of exposure 6

Sub-acute to chronic exposure

----AF for interspecies differences 1

Allometric scaling: rat versus human: the AF of 4 is already included in the route to route extrapolation

-----AF for intraspecies differences: general public 5

Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the subacute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces. As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.

----AF for other interspecies differences 1

The rats tolerated the dosing up to and including the limit dose 1000 mg/kg bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the fecesand no additional factorfor potential toxico-dynamicalterationin different animal species is justified.

----AF for quality of the whole database 1

There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a sub-acute guideline study indicating that Nigrosin WLF has a low toxicological potential and and the starting point for the DNEL derivation is conservative

----AF for remaining differences 1

In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)

Overall factor : 30

General Public DNEL (long term, systemic for inhalation route) 29 mg/m³

2.) DNEL (Short-term, inhalation route – systemic effects)

There is no information available to evaluate acute inhalation toxicity. Therefore, short term inhalation cannot be assessed nor be allocated to a hazard band. In addition, as there is no inhalation study available using repeated exposure the long term DNEL for inhalation route was derived from the available subacute oral study in animals dosed up to and including 1000 mg/kg bw/day and which was tolerated without impairment. According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (inhalation, long term) to DNEL (inhalation ,short term) if no data for acute inhalation toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied.

Thus, the DNEL (inhalation route, short term, systemic) is about: 58 mg/m³

3) DNEL (Long-term, oral route and dermal route - systemic effects)

Considering dermal exposure route and taking into account ECHA guidance document No R8 Version 2, December 2010, no additional default factor has to be introduced when performing oral to dermal extrapolation because it can be assumed that dermal absorption will not be higher than oral absorption:

NOAEL (subacute, oral) = NOAEL (subacute, dermal) = 1000 mg/kg bw/day

Factors to be applied Justification

----AF for dose response relationship 1

There are no effects up to the limit dose

----AF for differences in duration of exposure 6

Subacute to chronic exposure:

----AF for interspecies differences 4

Allometric scaling: rat versus human

----AF for intraspecies differences: General public 5

Based on the consideration by ECETOC Report TR110, 2010. Furthermore, there are no toxic effects up to the limit dose in the sub-acute rat study. In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces.As mentioned in the REACH Guidance R8 the intraspecies factor should be applied considering that “[h]umans differ in sensitivity to toxic insult due to a multitude of biological factors such as genetic polymorphism affecting e.g. toxicokinetics/metabolism, age, gender, health status and nutritional status.” Since the sub-acute study in rats indicates that Nigrosin WLF is excreted via the feces and probably absorbed only to a small amount it is likely that toxicokinetics/metabolism play a minor role for Nigrosin WLF. Consequently the default factor will be lowered taking into account compound specific data.

----AF for other interspecies differences 1

The rats tolerated the dosing up to and including the limit dose 1000 mg/kg/bw. Consequently it is considered to be a conservative starting point for DNEL calculation and the real NOAEL in rats is probably much higher. Based on this highly conservative NOAEL in rats it is likely that also other animals tolerate the test item without any harm at this dose and no additional interspecies uncertainty factor is warranted.In addition, at clinical observations it was noted that the color of feces was changed (black) in cages of treatment groups, indicating that the compound might be mainly excreted without absorption via the feces and no additional factor for potential toxico-dynamic alteration in different animal species is justified

AF for quality of the whole database 1 There is information available to cover all relevant toxicological endpoints. The available studies are performed according to guideline and GLP. No adverse effects were observed in a guideline sub-acute toxicity study indicating that Nigrosin WLF has a low toxicological potential and the starting point for the DNEL derivation is conservative.

----AF for remaining differences 1

In an evaluation by ECETOC 2003 and 2010 it is considered that routine application of the factor 2.5 is scientifically unjustified as a default factor. The view is supported by data generated by ERASM project (Barke et al 2010)

Overall factor 120

General Public DNEL long term, systemic for oral route 8.33 mg/kg bw/day

General Public DNEL long-term, systemic for dermal route 8.33 mg/kg bw/day

4.) DNEL (Short-term. oral route and dermal route – systemic effects)

There is an acute oral toxicity study in rats available resulting in a LD50>2000 mg/kg bw because the treatment was tolerated without impairment or mortality. Thus a NOAEL is not determined exactly.

With respect to dermal exposure, there are acute toxicity studies available using the dermal exposure route. These studies comprise acute toxicity, skin irritation and skin sensitization. In none of the three study types mortality or signs of intoxication were observed. The LD50 (dermal) is greater 2000 mg/kg bw and the test substance was not irritating to the skin nor caused skin sensitization. Thus, Nigrosin WLF cannot be allocated to a hazard band and a NOAEL for acute dermal toxicity is not determined exactely

Therefore it is proposed not to derive a DNEL for short term exposure by taking the derived DNEL (systemic, long term for oral and for dermal exposure route) as starting point. According to ECHA Guidance R8 a factor of 2-to 5 should be applied to extrapolate DNEL (long term) to DNEL ( short term) if no exact data for acute toxicity is available. As no relevant effects during subacute oral exposure are observed it is assumed that also short term exposure would be tolerated without impairment and therefore a factor of 2 is chosen to be applied.

Thus the DNEL (oral route, dermal route, short term, systemic) is about 16.7 mg/kg bw/day.

5.) DNELs (Reproductive toxicity -- Fertility Assessment)

There is no specific investigation available to evaluate the effects on fertility. In the available subacute toxicity study with oral application of doses up to and including 1000 mg/kg bw/day over a period of 28 days no adverse effects on reproductive organs are reported. Therefore, it is assumed that fertility is not affected by treatment with Nigrosin WLF and the NOAEL for fertility assessment complies with the NOAEL for general toxicity. Based on this consideration no additional DNEL has to be derived:

6.) DNELs (Reproductive toxicity -- Assessment for Development)

In a developmental toxicity study according OECD TG 414 the reproductive parameters (number of implantation sites, number of resorptions and number of fetuses) were not influenced by the test item. No dead fetuses and no test item-related malformations, variations or retardations were noted. Under the conditions of the study, Nigrosin WLF did not show any teratogenic potential.

The no-observed-adverse effect level (NOAEL) for the fetal organism was above 1000 mg Nigrosin WLF/kg bw/day.

III. Derivation of DNELs local

Basis for delineation of the DNELs (local)

According to ETAD Sub-komitee für Toxikologie: Methoden für toxikolog. Untersuchungen von Farbstoffen und Hilfsmitteln. Nigrosin WLF was tested for skin irritation and for irritation of the mucous membranes of the eyes. No irritation effects were observed neither to the skin of rabbits nor in the mucous membranes of the eyes.

Based on these results, no DNELs (local) need to be derived.´

Furthermore, the modified Local Lymph Node Assay (IMDS) was performed according to OECD Guidelines No. 429 and No. 406. The results show that there is no indication for a skin sensitizing effect after administration of a concentration up to and including 50 % Nigrosin WLF in this test system. No effects are observed.

IV: Conclusion (systemic and local effects)

The systemic DNELs for long term exposure include the short term hazard.

Therefore, the relevant DNELs are

General Public DNEL (systemic, inhalation route): 29 mg/m³

General Public DNEL (systemic, oral and dermal route): 8.33 mg/kg bw/day

With respect to local exposure no DNELs are needed because no hazard was identified.