Benzenamine, reaction products with aniline hydrochloride and nitrobenzene

Toxicological Summary

Workers - Hazard via inhalation route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

10.58 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):

25

Dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Modified dose descriptor starting point:

NOAEC

Value:

264 mg/m³

Explanation for the modification of the dose descriptor starting point:

As a point of departure for the inhalation long term DNEL, the NOAEL of 300 mg/kg bw/day from the 90-day repeated dose toxicity study in rats was chosen. In the absence of any experimental absorption data for both the starting route (oral) and the end route (inhalation), the worst case assumption was made, assuming a limited absorption for the oral route, leading to a low (conservative) internal NOAEL. It is proposed, in the absence of route-specific information on the starting route, to include a default factor of 2 (50% absorption is assumed for oral absorption, and 100% for inhalation.) in accordance with ECHA guidance document R.8 (ECHA, 2012).

 

The corrected dose descriptor for inhalation for workers = [300 mg/kg bw/day] / [0.38 m³/kg bw/day] × [6.7 m³/10m³] × (50% /100%) =264.47 mg/m³

AF for dose response relationship:

1

Justification:

Default value where the starting point is a clear NOAEL

AF for differences in duration of exposure:

2

Justification:

Subchronic to chronic

AF for interspecies differences (allometric scaling):

1

Justification:

N/A for inhalation

AF for other interspecies differences:

1

Justification:

Addressed by modification of starting point

AF for intraspecies differences:

5

Justification:

Default value for workers

AF for the quality of the whole database:

1

Justification:

Data available on the test substance appropriate for the tonnage band

AF for remaining uncertainties:

2.5

Justification:

Default value

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

DNEL related information

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:

3 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):

100

Dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Modified dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Explanation for the modification of the dose descriptor starting point:

As a point of departure for the dermal long term DNEL, the NOAEL of 300 mg/kg bw/day from the 90-day repeated dose toxicity study in rats was chosen. In the absence of data, 100% oral absorption is assumed and no additional correction factor was introduced.

AF for dose response relationship:

1

Justification:

The starting point is a NOAEL

AF for differences in duration of exposure:

2

Justification:

Subchronic to chronic

AF for interspecies differences (allometric scaling):

4

Justification:

Allometric scaling - rat to human

AF for other interspecies differences:

1

Justification:

Addressed by modification of starting point

AF for intraspecies differences:

5

Justification:

Default value for workers

AF for the quality of the whole database:

1

Justification:

Data available on the test substance appropriate for the tonnage band

AF for remaining uncertainties:

2.5

Justification:

Default value

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

DNEL related information

Local effects

Long term exposure

Hazard assessment conclusion:

no hazard identified

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

Workers - Hazard for the eyes

Local effects

Hazard assessment conclusion:

no hazard identified

Additional information - workers

Rationale for a key study selection for DNELs for Nigrosine

Key study selection has been undertaken in accordance with ECHA guidance (1).   The proposed NOAEL from the 28-day repeated dose toxicity study in Sprague-Dawley rats (Wragg and Brooks, 1994) and the 90-day repeated dose toxicity study in Wistar rats (Oroszlány, 2019) are primarily based on effects that are indicative of haemolytic anaemia and methaemoglobinaemia at 1000 mg/kg bw/day. The reported NOAEL is 150 mg/kg bw/day and 300 mg/kg bw/day for 28-day repeated dose toxicity study and the 90-day repeated dose toxicity study, respectively. The differences in NOAEL between the studies are due to the different intermediate dose levels employed in each study. No clear indications of haemolytic anaemia were observed in the intermediate or low dose levels in these studies. There is currently no convincing evidence to suggest there are strain differences with respect to the severity of chemically-induced haemolytic anaemia in rats. Apart from the haemolytic anaemia, thers that showed statistically significant changes were within the range of the laboratories historical control data or only slightly outside the ranges. No parameters associated with haemolytic anaemia were affected up to 1000 mg/kg bw/day in a range-finding prenatal toxicity study in female Wistar rats (Tuyl, 2007a). No adverse toxicity was seen in the other available studies up to the highest dose levels of 1000 mg/kg bw/day (Wragg and Brooks, 1993; Namiki, 2007; Tuyl, 2007b); although none of these studies measured parameters relevant for haemolytic anaemia. There is no indication of any direct toxicity of Nigrosine to the bone marrow that may be associated with the observed destruction of the erythrocytes and the increased burden to the spleen in any of these studies.

The NOAEL obtained from the 90-day repeated dose toxicity study in Wistar rats (i.e. 300 mg/kg bw/day) is considered to be the most suitable endpoint for DNEL derivations for the human health risk assessment for Nigrosine. The study was performed with the longest duration of exposure period among all the available studies with the repeated dose study regimens for the registered substance and has greater statistical power than the 28-day repeated dose toxicity study due to the higher number of animals employed in each group. Furthermore, the NOAEL from the 90-day repeated dose toxicity study is considered to more adequately cover the relevant exposure duration compared to the 28-day repeated dose toxicity for the effect of concern, given the red cell life span in rat is 50 days (Muller et al., 2006 (2)). Therefore, it is considered that the 90-day repeat dose study is the most appropriate study to use to control the long-term and short-term risks for the substance.

The dose level for the treatment-related systemic effects seen at 300 mg/kg bw/day exceed the cut-off dose of 100 mg/kg bw/day for STOT RE Cat 2, therefore the results are not subject to the classification for specific target organs.

Summary of relevant data

Study (Reference)	TG	Route of administration	Rat strain	No. of animals	Dose levels (mg/kg bw/day)	Observations
14-day repeated dose toxicity study in rats (Wragg and Brooks, 1993)	N/A	Gavage	Sprague-Dawley	3/sex/dose	0, 150, 400, 1000 (arachis oil)	Dark faeces from Day 3 in all dosed animals ↓bw gain in males and females at 1000 mg/kg bw/day Abnormally pink adipose tissue at ≥ 150 mg/kg bw/day, non-glandular gastric region with stained black at ≥ 150 mg/kg bw/day
A range-finding study in female rat (GD6-19) (Tuyl, 2007a). Dosing Days 6-19post-coitum	N/A	Gavage	Wistar	6/female/dose	0, 15, 150, 1000 (propylene glycol)	Reddish discolouration of the pancreatic or uterine adipose tissue at ≥ 150 mg/kg bw/day
28-day sub-acute oral (gavage) toxicity study in the rat (Wragg and Brooks, 1994)	407	Gavage	Sprague-Dawley	5/sex/dose	0, 15, 150, 1000 (arachis oil)	↓Hb, ↓RBC, ↓Hct, ↑MCV, ↑Meth in males and females at 1000 mg/kg bw/day ↑Retic, ↓MCHC, ↑MCH in females at 1000 mg/kg bw/day Abnormally pink adipose tissue at ≥ 150 mg/kg bw/day ↑absolute and relative to bw spleen weight in males and females at 1000 mg/kg bw/day, relative liver weight in females at 1000 mg/kg bw/day Periportal hepatocyte basophilia in males and females at 1000 mg/kg bw/day ↑ splenic extramedullary haemopoiesis and haemosiderin pigment deposition in males and females at 1000 mg/kg bw/day
Preliminary reproduction toxicity screening study of Nigrosine base Sapl by oral administration in rats (Namiki, 2007)	421	Gavage	Sprague-Dawley	12/sex/dose	0, 15, 150, 1000 (arachis oil)	Blackish faeces in all dosed animals at ≥ 15 mg/kg bw/day Abnormal contents in the stomach, blackish patch in the anterior stomach mucosa and pinkish patch in the adipose tissue in all dosed animals at ≥ 150 mg/kg bw/day (P & F1)
A prenatal developmental toxicity study of C.I. Solvent Black 7 in rats by oral gavage (Tuyl, 2007b)	414	Gavage	Wistar	24-25/female/dose	0, 15, 150, 1000 (propylene glycol)	Black discolouration/staining of faeces and different parts of the body ≥ 15 mg/kg bw/day Slight ↑Retic at 1000 mg/kg bw/day. Due to the absence of other effects in erythrocyte parameters this was considered of doubtful toxicological relevance Reddish discolouration in parts of the abdominal fat
Nubian black TN-870: a 90-day oral (gavage) toxicity study in Wistar rats(Oroszlány, 2019)	408	Gavage	Wistar	10/sex/dose	0, 100, 300, 1000 (corn oil)	↓RBC, ↓Hb, ↑Retic in males and females ≥ at 300 mg/kg bw/day (with statistical significance attained at 1000 mg/kg bw/day) ↑K^+in males at ≥ at 300 mg/kg bw/day, ↑T-Bil in females at 1000 mg/kg bw/day, ↑bile acids in males at 1000 mg/kg bw/day ↑ spleen weight (absolute, relative to bw and relative to brain weight in females; relative to bw in males) at 1000 mg/kg bw/dayg bw/day Brownish black faeces in all animals from Day2/3 until the end of treatment Pink discoloration of adipose tissue at in males and females at ≥ 100 mg/kg bw/day Black digestive content in the stomach at ≥ 100 mg/kg bw/day Grey mesenteric lymph node in males at 1000 mg/kg bw/day Extramedullary haematopoiesis in males and females at 1000 mg/kg bw/day

References:

(1)ECHA (2012) Guidance on information requirements and chemical safety assessment Guidance on information requirements and chemical safety assessment version: 2.1.

(2)Muller, A.M., Jacobsen, H., Healy, E.et al (2006) Hazard classification of chemicals inducing haemolytic anaemia: An EU regulatory perspective.Regulatory Toxicology and Pharmacology45(3): 229-241.

General Population - Hazard via inhalation route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

2.61 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):

50

Dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Modified dose descriptor starting point:

NOAEC

Value:

130.43 mg/m³

Explanation for the modification of the dose descriptor starting point:

As a point of departure for the inhalation long term DNEL, the NOAEL of 300 mg/kg bw/day from the 90-day repeated dose toxicity study in rats was chosen. In the absence of any experimental absorption data for both the starting route (oral) and the end route (inhalation), the worst case assumption was made, assuming a limited absorption for the oral route, leading to a low (conservative) internal NOAEL. It is proposed, in the absence of route-specific information on the starting route, to include a default factor of 2 (50% absorption is assumed for oral absorption, and 100% for inhalation.) in accordance with ECHA guidance document R.8 (ECHA, 2012).

 

The corrected dose descriptor for inhalation for general population = [300 mg/kg bw/day] / [1.15 m³/kg bw/day] × (50% /100%) =130.43 mg/m³

AF for dose response relationship:

1

Justification:

The starting point is a clear NOAEL

AF for differences in duration of exposure:

2

Justification:

Subchronic to chronic

AF for interspecies differences (allometric scaling):

1

Justification:

Allometric scaling - N/A for inhalation

AF for other interspecies differences:

1

Justification:

Addressed by modification of starting point

AF for intraspecies differences:

10

Justification:

Default value for general population

AF for the quality of the whole database:

1

Justification:

Data available on the test substance appropriate for the tonnage band

AF for remaining uncertainties:

2.5

Justification:

Default value

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

DNEL related information

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:

1.5 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):

200

Dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Modified dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Explanation for the modification of the dose descriptor starting point:

As a point of departure for the dermal long term DNEL, the NOAEL of 300 mg/kg bw/day from the 90-day repeated dose toxicity study in rats was chosen. A maximal absorption was already occurred by the oral route and no additional correction factor was introduced.

AF for dose response relationship:

1

Justification:

The starting point is a NOAEL

AF for differences in duration of exposure:

2

Justification:

Subchronic to chronic

AF for interspecies differences (allometric scaling):

4

Justification:

Allometric scaling - Rat to human

AF for other interspecies differences:

1

Justification:

Addressed by modification of starting point

AF for intraspecies differences:

10

Justification:

Default value for general population

AF for the quality of the whole database:

1

Justification:

Data available on the test substance appropriate for the tonnage band

AF for remaining uncertainties:

2.5

Justification:

Default value

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

DNEL related information

Local effects

Long term exposure

Hazard assessment conclusion:

no hazard identified

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

General Population - Hazard via oral route

Systemic effects

Long term exposure

Hazard assessment conclusion:

DNEL (Derived No Effect Level)

Value:

1.5 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):

200

Dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Modified dose descriptor starting point:

NOAEL

Value:

300 mg/kg bw/day

Explanation for the modification of the dose descriptor starting point:

No modification of the dose descriptor starting point is considered necessary.

AF for dose response relationship:

1

Justification:

The starting point is a NOAEL

AF for differences in duration of exposure:

2

Justification:

Subchronic to chronic

AF for interspecies differences (allometric scaling):

4

Justification:

Allometric scaling - Rat to human

AF for other interspecies differences:

1

Justification:

Default value - considered in modification of starting point

AF for intraspecies differences:

10

Justification:

Default value for general population

AF for the quality of the whole database:

1

Justification:

Data available on the test substance appropriate for the tonnage band

AF for remaining uncertainties:

2.5

Justification:

Default value

Acute/short term exposure

Hazard assessment conclusion:

no hazard identified

DNEL related information

General Population - Hazard for the eyes

Local effects

Hazard assessment conclusion:

no hazard identified

Additional information - General Population

Rationale for a key study selection for DNELs for Nigrosine

Key study selection has been undertaken in accordance with ECHA guidance (1).   The proposed NOAEL from the 28-day repeated dose toxicity study in Sprague-Dawley rats (Wragg and Brooks, 1994) and the 90-day repeated dose toxicity study in Wistar rats (Oroszlány, 2019) are primarily based on effects that are indicative of haemolytic anaemia and methaemoglobinaemia at 1000 mg/kg bw/day. The reported NOAEL is 150 mg/kg bw/day and 300 mg/kg bw/day for 28-day repeated dose toxicity study and the 90-day repeated dose toxicity study, respectively. The differences in NOAEL between the studies are due to the different intermediate dose levels employed in each study. No clear indications of haemolytic anaemia were observed in the intermediate or low dose levels in these studies. There is currently no convincing evidence to suggest there are strain differences with respect to the severity of chemically-induced haemolytic anaemia in rats. Apart from the haemolytic anaemia, thers that showed statistically significant changes were within the range of the laboratories historical control data or only slightly outside the ranges. No parameters associated with haemolytic anaemia were affected up to 1000 mg/kg bw/day in a range-finding prenatal toxicity study in female Wistar rats (Tuyl, 2007a). No adverse toxicity was seen in the other available studies up to the highest dose levels of 1000 mg/kg bw/day (Wragg and Brooks, 1993; Namiki, 2007; Tuyl, 2007b); although none of these studies measured parameters relevant for haemolytic anaemia. There is no indication of any direct toxicity of Nigrosine to the bone marrow that may be associated with the observed destruction of the erythrocytes and the increased burden to the spleen in any of these studies.

The NOAEL obtained from the 90-day repeated dose toxicity study in Wistar rats (i.e. 300 mg/kg bw/day) is considered to be the most suitable endpoint for DNEL derivations for the human health risk assessment for Nigrosine. The study was performed with the longest duration of exposure period among all the available studies with the repeated dose study regimens for the registered substance and has greater statistical power than the 28-day repeated dose toxicity study due to the higher number of animals employed in each group. Furthermore, the NOAEL from the 90-day repeated dose toxicity study is considered to more adequately cover the relevant exposure duration compared to the 28-day repeated dose toxicity for the effect of concern, given the red cell life span in rat is 50 days (Muller et al., 2006 (2)). Therefore, it is considered that the 90-day repeat dose study is the most appropriate study to use to control the long-term and short-term risks for the substance.

The dose level for the treatment-related systemic effects seen at 300 mg/kg bw/day exceed the cut-off dose of 100 mg/kg bw/day for STOT RE Cat 2, therefore the results are not subject to the classification for specific target organs.

Summary of relevant data

Study (Reference)	TG	Route of administration	Rat strain	No. of animals	Dose levels (mg/kg bw/day)	Observations
14-day repeated dose toxicity study in rats (Wragg and Brooks, 1993)	N/A	Gavage	Sprague-Dawley	3/sex/dose	0, 150, 400, 1000 (arachis oil)	Dark faeces from Day 3 in all dosed animals ↓bw gain in males and females at 1000 mg/kg bw/day Abnormally pink adipose tissue at ≥ 150 mg/kg bw/day, non-glandular gastric region with stained black at ≥ 150 mg/kg bw/day
A range-finding study in female rat (GD6-19) (Tuyl, 2007a). Dosing Days 6-19post-coitum	N/A	Gavage	Wistar	6/female/dose	0, 15, 150, 1000 (propylene glycol)	Reddish discolouration of the pancreatic or uterine adipose tissue at ≥ 150 mg/kg bw/day
28-day sub-acute oral (gavage) toxicity study in the rat (Wragg and Brooks, 1994)	407	Gavage	Sprague-Dawley	5/sex/dose	0, 15, 150, 1000 (arachis oil)	↓Hb, ↓RBC, ↓Hct, ↑MCV, ↑Meth in males and females at 1000 mg/kg bw/day ↑Retic, ↓MCHC, ↑MCH in females at 1000 mg/kg bw/day Abnormally pink adipose tissue at ≥ 150 mg/kg bw/day ↑absolute and relative to bw spleen weight in males and females at 1000 mg/kg bw/day, relative liver weight in females at 1000 mg/kg bw/day Periportal hepatocyte basophilia in males and females at 1000 mg/kg bw/day ↑ splenic extramedullary haemopoiesis and haemosiderin pigment deposition in males and females at 1000 mg/kg bw/day
Preliminary reproduction toxicity screening study of Nigrosine base Sapl by oral administration in rats (Namiki, 2007)	421	Gavage	Sprague-Dawley	12/sex/dose	0, 15, 150, 1000 (arachis oil)	Blackish faeces in all dosed animals at ≥ 15 mg/kg bw/day Abnormal contents in the stomach, blackish patch in the anterior stomach mucosa and pinkish patch in the adipose tissue in all dosed animals at ≥ 150 mg/kg bw/day (P & F1)
A prenatal developmental toxicity study of C.I. Solvent Black 7 in rats by oral gavage (Tuyl, 2007b)	414	Gavage	Wistar	24-25/female/dose	0, 15, 150, 1000 (propylene glycol)	Black discolouration/staining of faeces and different parts of the body ≥ 15 mg/kg bw/day Slight ↑Retic at 1000 mg/kg bw/day. Due to the absence of other effects in erythrocyte parameters this was considered of doubtful toxicological relevance Reddish discolouration in parts of the abdominal fat
Nubian black TN-870: a 90-day oral (gavage) toxicity study in Wistar rats(Oroszlány, 2019)	408	Gavage	Wistar	10/sex/dose	0, 100, 300, 1000 (corn oil)	↓RBC, ↓Hb, ↑Retic in males and females ≥ at 300 mg/kg bw/day (with statistical significance attained at 1000 mg/kg bw/day) ↑K^+in males at ≥ at 300 mg/kg bw/day, ↑T-Bil in females at 1000 mg/kg bw/day, ↑bile acids in males at 1000 mg/kg bw/day ↑ spleen weight (absolute, relative to bw and relative to brain weight in females; relative to bw in males) at 1000 mg/kg bw/dayg bw/day Brownish black faeces in all animals from Day2/3 until the end of treatment Pink discoloration of adipose tissue at in males and females at ≥ 100 mg/kg bw/day Black digestive content in the stomach at ≥ 100 mg/kg bw/day Grey mesenteric lymph node in males at 1000 mg/kg bw/day Extramedullary haematopoiesis in males and females at 1000 mg/kg bw/day

References

(1) ECHA (2012) Guidance on information requirements and chemical safety assessment Guidance on information requirements and chemical safety assessment version: 2.1.

(2) Muller, A.M., Jacobsen, H., Healy, E.et al (2006) Hazard classification of chemicals inducing haemolytic anaemia: An EU regulatory perspective.Regulatory Toxicology and Pharmacology45(3): 229-241.