N-1-naphthylaniline

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

basic toxicokinetics in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

distribution

excretion

metabolism

toxicokinetics

Principles of method if other than guideline:

- Kinetic determination of plasma and tissue radioactivity of rats following an oral dose of 14C-PNA.
- Determination of radioactivity in rat tissues 24 hours after administration of an oral dose of 14C-PNA.
- Radiolabeled PNA and metabolites were determined in urine samples pooled over 48 hours.
- Excretion was determined by investigation of total radioactivity in urine and feces of rats after various intervals of time following application of 14C-PNA.

GLP compliance:

not specified

Radiolabelling:

yes

Remarks:

14C

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, New York
- Weight at study initiation: 225 - 250 g
- Acclimation period: at least 1 week


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

single exposure

Dose / conc.:

160 mg/kg bw/day

No. of animals per sex per dose / concentration:

- Kinetics and Tissue distribution: 3 males per observation interval
- Metabolism and excretion: not specified

Control animals:

not specified

Details on study design:

no information given

Details on dosing and sampling:

PHARMACOKINETIC STUDY (Absorption, distribution, excretion)
- Tissues and body fluids sampled (Plasma kinetics): blood, plasma
- Tissues and body fluids sampled (Tissue distribution): heart, lung, pancreas, spleen, kidney, testes, liver, fat, stomach, small intestine and contents, caeca, large intestine and contents
- Tissues and body fluids sampled (Excretion): urine, feces
- Time and frequency of sampling (Plasma kinetics): 0.5, 1, 2, 4, 8, 12, 16, 24, 48 and 72 hours
- Time and frequency of sampling (Tissue distribution): 24 hours; for tissue kinetics: 0.5, 1, 2, 4, 8, 12, 16, 24 and 48 hours
- Time and frequency of sampling (Excretion): 2, 4, 8, 12, 24, 32, 48, 72 and 120 hours

METABOLITE CHARACTERISATION STUDIES
- Tissues and body fluids sampled: urine
- Time and frequency of sampling: pooled over 48 hours
- From how many animals: no data on animal numbers, no data if individual samples were pooled, individual samples were pooled over sampling period
- Method type(s) for identification: HPLC-UV, Liquid scintillation counting
- Limits of detection and quantification: no data

Statistics:

Percentage of radioactivity in urine extracts.

Details on distribution in tissues:

Kinetics: the concentration of total radioactivity in the plasma reached a peak of 116 µg PNA equivalents/ml four hours after dosing, thereafter it declined in a biphasic manner. About 3 % of the radioactivity in the plasma could be attributed to the parent compound 4 hours after dosing. The appearance and disappearance of plasma radioactivity shown was fitted to a pharmacokinetic two-compartment open-system model, in which a central blood compartment is in reversible equilibrium with a peripheral tissue compartment, with first-order-absorption. Analysis of the plasma radioactivity according to this relationship gave half-life values of 1.59 hr for the appearance process and 1.68 and 33 hr, respectively, for the two elimination processes. The level of unchanged PNA in the plasma followed a kinetic pattern similar to that of the total 14C but reached its peak level off 1.25 µg PNA/ml plasma at 2 hr after dosing, after which it declined in a biphasic manner. Analysis of the plasma levels of PNA gave half-life values of 0.66 hr for the appearance process and 1.24 and 11.1 hr, respectively, for the two disappearance processes.

Tissue distribution: Appreciable radioactivity was detected in all tissues at 24 hours indicating a rapid absorption and distribution of 14C-PNA and its metabolites. The major sites of distribution of 14C were fat, liver, kidney and lungs with large amounts in the gastro-intestinal tract plus contents. In all four major tissues (liver, fat, kidney, lung), radioactivity reached a maximum about 4 hours after dosing, reflecting a rapid uptake and distribution of the chemical. The levels of radioactivity then appeared to decrease in a biphasic manner. At all times, the highest concentration of 14C was found in the liver followed by fat, kidney, and lung. When radioactivity is expressed in terms of percent of the total dose received, only liver and fat are important tissues which retained most of the unexcreted radioactivity.

Details on excretion:

More than 90 % of the administered dose had been excreted into the urine and feces within 48 hours of administration. The primary route of excretion of radioactivity was via the feces, although appreciable amounts of 14C were excreted via the urine. After 72 hours, 60 % of the total dose had been excreted in the feces and about 35 % in the urine. The bulk of the excretion in urine and feces occurred during the first 24 hours after administering the chemical.

Metabolites identified:

yes

Details on metabolites:

Urine contained a high percentage of 14C as materials which remained in the aqueous phase following extraction at pH 12 and 2, suggesting that PNA was extensively metabolized by the rat. HPLC analysis of the ether extract of the urine showed at least five 14C-metabolites (see table), no PNA could be detected in the ether extracts.

Tissue distribution

Distribution of radioactivity in rat tissues 24 hours after oral administration of¹⁴C-PNA:

Tissue	^14C-PNA Equivalent µg/g wet tissue	Percent of administered dose
Heart	6.49	0.0130
Lung	10.2	0.0280
Pancreas	3.62	0.00432
Spleen	3.18	0.00377
Kidney	28.7	0.123
Testes	3.30	0.0200
Liver	33.8	0.415
Fat	66.0	2.35
Stomach	488	5.62
Small intestine (+ contents)	235	5.34
Caeca	362	5.67
Large intestine (+ contents)	435	3.10

Metabolism

Distribution of radioactivity in 0-48 hr pooled urine samples:

Fraction	Percent of total radioactivity in the urine
Ether extract (pH 12)	16
Ether extract (pH 2)	19
Aqueous residue (unextractable)	65

Retention volumes and relative abundance of¹⁴C-PNA metabolites in the ether extract (pH 2):

^14C-Compound	HPLC Retention Volume (ml)	% of^14C in the Extract
Area A           1	2.3	14
2	3.4	20
3	4.2	11
4	5.8
5	7.5
6	9.0	Σ 18
Area B	12.0	23
Area C	14.5	14
PNA	28.0	0

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Principles of method if other than guideline:

Identification of PNA-metabolites using rat liver microsomes from Aroclor 1254-induced rats.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, New York
- Weight at study initiation: 225 - 250 g
- Acclimation period: at least 1 week


ENVIRONMENTAL CONDITIONS
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

other: not applicable, in vitro

Vehicle:

DMSO

Duration and frequency of treatment / exposure:

3 hours at 37°C

Dose / conc.:

3.8 other: mg PNA/100mL

No. of animals per sex per dose / concentration:

not applicable, in vitro

Control animals:

other: not applicable

Metabolites identified:

yes

Details on metabolites:

Several metabolites were identfied: mono- and di-hydroxylated derivatives of PNA and the corresponding quinone derivatives, indicating further facile air oxidation of the metabolites during handling. In the Mono-hydroxy derivative, the hydroxy group is in the naphtalene moiety at the position para to the amino group. In the dihydroxy derivative, at least one hydroxy group is at the available para position in the naphtyl ring. Concerning the other hydroxy group the authors favour the idea of a localisation at the para position in the phenyl ring, although the existence of a hydroxy group in each ring of the naphtalene moiety cannot be excluded.

Reference 3

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Objective of study:

metabolism

Principles of method if other than guideline:

The induction of metabolism was investigated in liver microsomes from rats either with or without pre-treatment with metabolic inducers or the test substance itself. The metabolites formed out of the test substance were identified in experiments with freshly isolated rat hepatocytes.

GLP compliance:

not specified

Radiolabelling:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Neuherberg, GSF Research Center Ger., inbred strain)

Route of administration:

other: pre-treatment by intraperitoneal injection or oral gavage

Vehicle:

olive oil

Duration and frequency of treatment / exposure:

Pre-treatment: single i.p. injection, or repeatedly for 4 days or 8 days/3 weeks by oral gavage

Dose / conc.:

200 other: mg/kg bw

Remarks:

i.p.

Dose / conc.:

500 other: mg/kg bw

Remarks:

i.p.

Dose / conc.:

1 000 other: mg/kg bw

Remarks:

i.p.

Dose / conc.:

200 mg/kg bw/day

Remarks:

gavage

No. of animals per sex per dose / concentration:

no data

Control animals:

yes, concurrent vehicle

Positive control reference chemical:

Pre-treatment with 3-methylcholanthrene (3-MC), 20 mg/kg bw/day on 2 consecutive days; or phenobarbital (PB), single i.p. injection of 80 mg/kg bw in saline, followed by 0.1 % PB in the drinking water for 1 week.

Details on dosing and sampling:

not applicable, in vitro

Statistics:

no data

Metabolites identified:

yes

Details on metabolites:

One major phenolic metabolite of PANA was identified in experiments with rat hepatocytes, very likely 4-hydroxy-PANA, but the identity could not be confirmed completely. The metabolite was formed by cytochrome P-450-dependent monooxygenases via the epoxide pathway, as demonstrated in the experiments with rat liver microsomes pre-treatment with 3-MC and PB. Furthermore, pre-treatment of rats with PANA was also able to induce microsomal CYP450-dependent monooxygenases. No formation of 1-naphtylamine was detected.

Relative rates of microsomal metabolism of PANA by various treatments:

Pre-treatment	Dose (mg/kg)	No. of treatment	Sex	Rate, nmoles/min/mg protein
Control	--	--	Male	0.18 ± 0.03
PB	40	1	Male	0.29 ± 0.02*
MC	20	2	Male	0.30 ± 0.04*
Control	--	--	Male	0.42 ± 0.01
PANA	200	4	Male	0.37 ± 0.04
Control	olive oil	--	Female	0.24 ± 0.10
PANA	200	8^a	Female	0.60 ± 0.10*

The data are mean of four single determination ± SD.

*P<0.05

^a3 days/week for female rats by gastric intubation.

Effect of animal pre-treatment with inducers of monooxygenase activity:

					Rates, nmoles/min/mg protein
Pre-treatment	Dose (mg/kg)	No. of treatment	Sex	Cytochrome P-450 (nmoles/mg protein)	Ethylmorphine demethylation	Aldrin epoxydation	Benzo[a]pyrene hydroxylation
Control	--	--	Male	0.72 ± 0.08	7.40 ± 1.13	2.88 ± 0.36	0.24 ± 0.02
PB	40	1	Male	1.50 ± 0.11*	14.20 ± 2.20	5.18 ± 1.00	0.42 ± 0.03*
MC	20	2	Male	0.84 ± 0.07	7.20 ± 0.70	2.52 ± 0.40	0.76 ± 0.13*
Control	--	--	Male	0.89 ± 0.05	5.65 ± 0.63	2.82 ± 0.21	0.24 ± 0.01
PANA	200	1	Male	0.95 ± 0.06	8.50 ± 1.60	3.10 ± 0.16	0.22 ± 0.03
	500	1	Male	1.12 ± 0.05*	11.90 ± 0.13**	4.54 ± 0.07**	0.25 ± 0.04
	1000	1	Male	1.25 ± 0.03*	12.20 ± 0.80**	4.39 ± 0.20*	0.23 ± 0.02
Control	--	--	Male	0.87 ± 0.04	8.61 ± 0.69	7.48 ± 0.47	0.22 ± 0.04
P1NA	200	4	Male	1.03 ± 0.03*	13.41 ± 1.56	5.38 ± 0.45	0.30 ± 0.03
Control	olive oil	--	Female	0.90 ± 0.12	0.71 ± 0.20	0.27 ± 0.04	0.02 ± 0.01
PANA	200	8^a	Female	0.92 ± 0.096	2.85 ± 0.41**	0.47 ± 0.08	0.10 ± 0.02**

Microsomes from male or female rats weighing 150 g were assayed. The data are mean of four single determinations ± SD.

*P<0.05; **P<0.01.

^a3 days/week by gastric intubation.

Reference 4

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Sep 2020 to 19 Mar 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Version / remarks:

Adopted: 13 April 2004

Deviations:

no

GLP compliance:

yes

Radiolabelling:

yes

Species:

other: Human Skin Donors

Type of coverage:

open

Vehicle:

other: neat compound, rubber matrix and base oil for lubricants

Duration of exposure:

The exposure period was terminated at 8 h after dosing. At 24 hours post dose, i.e. after 16 hours monitoring period, each diffusion cell was dismantled and the skin removed. See any other information on material and methods for further details.

Doses:

- Nominal doses: 1 % and 5 % in base oil for lubricants, 100 % as neat powder and 0.5 % and 3.5 % in rubber matrix
- Actual doses: 1.04 % (w/v), 5.34 % (w/v), 100 % (w/w), 0.409 % (w/w), 3.17 % (w/w)
- Actual doses calculated as follows: Seven representative weighed discs per dose were collected throughout dosing, seven representative aliquots of test preparation 1 and 2 (1 and 5% in base oil for lubricants) were dispensed into vials at the time of dosing and seven representative aliquots of the test preparation 3 (100% w/w) were collected immediately after dosing. All representative aliquots were analysed by liquid scintillation counting.
- Dose volume: 10 µL/cm² for 1% and 5% in base oil for lubricants, 5 mg/cm² for the 100% neat powder and 1 disc per skin sample for the 0.5% and 3.5% in rubber matrix (diameter of 20 mm and thickness 1120-1220 µm and 1150-1470 µm, respectively)
- Rationale for dose selection: in line with the ECHA final decision, several small dosages representative of the in-use conditions were chosen

No. of animals per group:

A total of 12 samples of human split thickness skin membranes obtained from four different donors were used per test preparation.

Details on study design:

DOSE PREPARATION
- Method for preparation of dose suspensions:
After assessment of the radiochemical purity the radiochemical was diluted with non-radiolabelled PANA using ethanol (which was evaporated afterwards). The concentration and homogeneity of the mixture was assessed by liquid scintillation counting.
For test preparation 1 and 2, radiolabelled PANA was incorporated into a base oil for lubricants at a concentration of approx. 1 and 5 %, respectively.
For test preparation 4 and 5, radiolabelled PANA at a concentration of 0.5 and 3.5 % incorporated into a rubber disc by homogeneous mixing into a rubber mix. After mixing, the compounds are pressed into 1 mm thick mixture skins and discs with a diameter on 20 mm are punched out. Rubber disc containing 0.5 % and 3.5 % PANA respectively were applied to the skin samples.

APPLICATION OF DOSE:
Test preparation 1 and 2 were applied evenly over the surface of the exposed skin of 12 split thickness samples of human skin using a positive displacement pipette set to deliver ca 31.4 µL (10 µL/cm²). For [phenyl U 14C]-PANA in Test Preparation 3, water (31.4 µL) was applied to wet the skin surface. Test Preparation 3 was weighed into weighing boats (ca 15.7 mg) then tapped onto the skin surface and distributed evenly using a glass rod (application rate of 5 mg/cm²)
For test preparation 4 and 5 the skin surface was wetted prior to dosing with water (10 µL/cm²) and a single disc was applied using tweezers to a minimum of 12 skin samples. A glass rod was used to press down the disc to the skin.

VEHICLE
- Justification for use and choice of vehicle: rubber matrix and base oil for lubricants, representative of in-use conditions

TEST SITE
- Preparation of test site: The glass static diffusion cells were placed in a manifold on a magnetic stirrer plate heated via a circulating water bath to maintain the skin surface temperature at 32°C ± 1°C. The actual cell temperatures (ranging from 31.1°C to 32.6°C) were calibrated prior to mounting the skin membranes.
Magnetic stirrer bars were placed in the receptor fluid chambers which were filled with receptor fluid. Once thawed, sections of split thickness skin (ca 3 x 3 cm) were cut and mounted in the diffusion cells between the donor and receptor chamber. The donor chamber was tightened into place with a clamp. Cells were visually checked to ensure no cells were leaking and no air bubbles were present in the receptor fluid chamber.

Skin samples were allowed to equilibrate at 32°C ± 1°C for a minimum of 5 min. PBS (3 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further minimum of 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root mean squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 4.0 kΩ was excluded from subsequent absorption measurements. The PBS was removed from the skin surface; the skin was rinsed with water and dried with a tissue swab.
- Area of exposure: The surface area of exposed skin within the cells was 3.14 cm² with a receptor chamber volume of ca 10 mL (nominal).

REMOVAL OF TEST SUBSTANCE
- Washing procedures and type of cleansing agent: Commercial hand wash soap (ca 50 µL) was applied to the skin and the soap gently rubbed onto the skin with a tissue swab. The skin was then rinsed with ca 5 mL of a ca 2% (v/v) commercial soap solution. The soap solution was applied in aliquots (1 mL) and each aliquot was aspirated three times with a pipette. The skin was dried with a tissue swab. The process was repeated and the skin was dried with an additional tissue swab.
- Time after start of exposure: The exposure period was terminated at 8 h after dosing.

SAMPLE COLLECTION
Prior to dosing, a 300 µL sample of receptor fluid was removed from the receptor chamber collection arm. The receptor fluid volume was then maintained by the addition of fresh receptor fluid up to the calibration line on the receptor chamber collection arm. Following sample collection, the receptor chamber collection arm was sealed with Parafilm® to prevent evaporation of receptor fluid.
Receptor fluid aliquots were collected at 1, 2, 4, 8 and 12 h post dose as described above. All receptor fluid samples were analysed by liquid scintillation counting.

- Terminal procedure:
At 24 h post dose, the cell were dismantled and the donor chamber were retained for analysis (donor chamber wash). Skin was removed from the cell and placed on a piece of tissue paper to remove any remaining receptor fluid from the underside of the skin. The tissue was placed in the receptor wash pot for that particular cell.
The stratum corneum was removed with 20 successive tape strips. The skin sample was rotated 90° after each tape strip. Rotation was stopped if the epidermis/dermis junction became fragile or if epidermis was removed. Each tape was analysed individually by liquid scintillation counting.
The skin under the cell flange (unexposed skin) was cut away from the exposed skin. Each exposed skin sample was wrapped in a piece of clingfilm. A ca 200 g weight was heated to ca 65°C in a water bath and placed onto the epidermal surface for ca 90 s. The epidermis was then separated from the dermis using a scalpel. The clingfilm was retained for analysis.
Bulk receptor fluid (ca 10 mL) was removed from each receptor chamber and retained for analysis.
The receptor chambers were rinsed with ethanol (40 mL). The solvent was pooled as a single sample into the pre weighed receptor wash pot and sample weights were taken.
Total radioactivity was analysed in all samples of skin, receptor fluid, and washes by liquid scintillation counting.

SAMPLE PREPARATION
- Storage procedure: All samples were stored at ambient temperature for analysis. Following analysis, bulk samples were stored in a freezer set to maintain a temperature of -20°C.
- Preparation details: Skin samples were solubilised using Solvable® prior to analysis by liquid scintillation counting.

ANALYSIS
- Method type(s) for identification: All samples prepared in scintillation fluid were subjected to liquid scintillation counting, together with representative blank samples, using a Liquid Scintillation Analyser with automatic quench correction by an external standard method.
- Limits of detection and quantification: A limit of reliable measurement of 30 d.p.m. above background has been instituted

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: Eight donors were obtained from Tissue Solutions Ltd and two donors were obtained from BioIVT. The samples arrived at Charles River frozen and were stored in a freezer set to maintain a temperature of 20°C until used in the study.
- Donor information: 10 donors (male and female) aged 32 to 58 years old
- Ethical approval if human skin: Ethical approval for receipt and use of human skin has been obtained (Lothian Local Research Ethics Committee, REC Reference No. 06/S1101/19 and Glasgow Royal Infirmary REC, REC Reference No. 08/S0704/30). Tissue is regularly sourced from hospitals and tissue banks.
- Type of skin: full-thickness human skin (abdomen)
- Preparative technique: Human skin samples were removed from -20°C storage and allowed to thaw at ambient temperature. The thickness of the full thickness skin membranes was measured using a micrometer. Split-thickness membranes were prepared by pinning the full thickness skin, stratum corneum uppermost, onto a raised cork board and cutting with an electric dermatome (Zimmer®) at a setting equivalent to 200 400 µm depth. The thickness of the membranes was measured using a micrometer. Membranes were then wrapped in foil, placed into a self sealing bag and stored in a freezer, set to maintain a temperature of -20°C, for a maximum period of two months.
A static diffusion cell system (PermeGear Inc) was used.
- Thickness of skin (in mm): split-thickness range from 0.38 to 0.4 mm
- Membrane integrity check: Skin samples were allowed to equilibrate at 32°C ± 1°C for a minimum of 5 min. PBS (3 mL) was then added to the donor chamber and the skin samples were allowed to equilibrate for a further minimum of 30 min. The electrical resistance was then measured using a Tinsley Databridge (Model 6401) set at low voltage alternating current, 1000 Hz with a maximum voltage of 300 mV root mean squared (rms) in the parallel equivalent circuit mode. Any skin sample exhibiting a resistance less than 4.0 kΩ was excluded from subsequent absorption measurements. The PBS was removed from the skin surface; the skin was rinsed with water and dried with a tissue swab.
- Storage conditions: -20°C
- Justification of species, anatomical site and preparative technique: The human has been chosen for the safety evaluation as workers and consumers will be exposed during handling of the test item or the use of products containing the test item.


PRINCIPLES OF ASSAY
- Diffusion cell: A static cell diffusion system was used.
- Receptor fluid: Phosphate buffered saline (PBS) containing polyoxyethylene 20 oleyl ether (PEG, ca 6%, w/v), sodium azide (ca 0.01%, w/v), streptomycin (0.1 mg/mL) and penicillin (100 units/mL) with the pH confirmed and adjusted to 7.4 ± 0.1 if required.
- Solubility of test substance in receptor fluid: The target concentration (2.198 mg/mL) represented the maximum concentration of test item in the receptor fluid based on the entire applied dose being absorbed in 24 h from the 3.5% (w/w) rubber discs. As 83.78% of the target concentration was recovered, the receptor fluid was accepted for use on the study. The maximum absorption (equivalent to 5.11 µg/mL, Cell 5) was greatly below this level in the study, showing the receptor fluid was not rate limiting.
- Static system: The static diffusion cells was positioned in a steel manifold and heated via a circulating water bath to maintain a skin surface temperature
- Test temperature: 32 +/- 1 °C
- Occlusion: The donor chambers were not occluded and left open to the atmosphere

Signs and symptoms of toxicity:

no effects

Dermal irritation:

no effects

Absorption in different matrices:

- Skin wash:
Test preparation 1: 20.03 ± 9.14 % applied dose;
Test preparation 2: 30.28 ± 15.35 % applied dose;
Test preparation 3: 32.58 ± 7.60 % applied dose;
Test preparation 4: 0.07 ± 0.03 % applied dose;
Test preparation 5: 0.13 ± 0.10 % applied dose
- Skin test site:
Test preparation 1: 94.60 ± 3.24 % applied dose;
Test preparation 2: 93.84 ± 23.96 % applied dose;
Test preparation 3: 94.76 ± 1.49 % applied dose;
Test preparation 4: 98.05 ± 1.26 % applied dose;
Test preparation 5: 100.57 ± 1.50 % applied dose
- Skin, untreated site:
Test preparation 1: °0.40 ± °0.69 % applied dose;
Test preparation 2: °0.30 ± °0.88 % applied dose;
Test preparation 3: °0.01 ± °0.01 % applied dose;
Test preparation 4: °0.00 ± °0.00 % applied dose;
Test preparation 5: °0.00 ± °0.00 % applied dose
- Receptor fluid, receptor chamber, donor chamber (in vitro test system):
Receptor fluid:
Test preparation 1: 1.78 ± 0.85 % applied dose;
Test preparation 2: 1.66 ± 0.54 % applied dose;
Test preparation 3: 0.08 ± 0.04 % applied dose;
Test preparation 4: 0.22 ± 0.06 % applied dose;
Test preparation 5: 0.24 ± 0.07 % applied dose
Receptor chamber:
Test preparation 1: °0.28 ± °0.16 % applied dose;
Test preparation 2: 0.22 ± 0.17 % applied dose;
Test preparation 3: °0.02 ± °0.01 % applied dose;
Test preparation 4: °0.03 ± °0.01 % applied dose;
Test preparation 5: °0.03 ± °0.01 % applied dose
- Stratum corneum (in vitro test system):
Stratum Corneum 1-2:
Test preparation 1: 0.24 ± 0.20 % applied dose;
Test preparation 2: 0.37 ± 0.33 % applied dose;
Test preparation 3: 0.24 ± 0.18 % applied dose;
Test preparation 4: 0.01 ± 0.01 % applied dose;
Test preparation 5: 0.01 ± 0.01 % applied dose
Stratum Corneum 3-20:
Test preparation 1: 0.65 ± 0.44 % applied dose;
Test preparation 2: 1.14 ± 1.16 % applied dose;
Test preparation 3: 0.18 ± 0.11 % applied dose;
Test preparation 4: 0.02 ± 0.03 % applied dose;
Test preparation 5: 0.03 ± 0.02 % applied dose


°=Mean includes results calculated from data less than 30 d.p.m above background

Total recovery:

- Total recovery:
Test preparation 1: 100.49 ± 1.39 % applied dose;
Test preparation 2: 100.16 ± 23.47 % applied dose;
Test preparation 3: 95.96 ± 1.30 % applied dose;
Test preparation 4: 98.38 ± 1.24 % applied dose;
Test preparation 5: 100.93 ± 1.51 % applied dose
- Recovery of applied dose acceptable: Mass balance should be within 85-115%. However, if the mass balance was below 85% and the loss can be explained, the samples may also be accepted.
- Results adjusted for incomplete recovery of the applied dose:
The mass balance for most individual samples was within 100 ± 15% except for 4 samples for test preparation 2: Cell 37 (137.01%), Cell 38 (55.79%), Cell 39 (138.06%) and Cell 40 (73.04%). IncIt is thought that an error occurred during the wash for this study, as if the tissue swab values were swapped with the cells’ donor pair mass balance for each cell would be acceptable. Incorrect dosing has also been excluded as the same pipette and pipette setting were used throughout dosing. For these reasons, cells have been included in calculations..
- Limit of detection (LOD): A limit of reliable measurement of 30 d.p.m. above background has been instituted in these laboratories.
- Quantification of values below LOD or LOQ: Counts that are below 30 d.p.m. above background represent a true value. This means that data are recorded with values that are less than the limit of reliable measurement and marked.

Time point:

24 h

Dose:

1%

Parameter:

percentage

Absorption:

5.6 %

Time point:

24 h

Dose:

5%

Parameter:

percentage

Absorption:

6.4 %

Time point:

24 h

Dose:

100%

Parameter:

percentage

Absorption:

0.66 %

Time point:

24 h

Dose:

0.5%

Parameter:

percentage

Absorption:

0.35 %

Time point:

24 h

Dose:

3.5%

Parameter:

percentage

Absorption:

0.38 %

Conversion factor human vs. animal skin:

not applicable

Conclusions:

Following topical application of [phenyl U 14C]PANA to human skin in vitro dermal absorption after accounting for variability between replicates is 0.38% (17.6 μg equiv./cm²) of the applied dose in rubber discs (ca 3.5%, w/w), 0.35% (1.91 μg equiv./cm²) of the applied dose in rubber discs (ca 0.5%, w/w), 5.6% (5.84 μg equiv./cm²) of the applied dose in Test Preparation 1 (ca 1%, w/v), 6.4% (34.4 μg equiv./cm²) of the applied dose in Test Preparation 2 (ca 5%, w/v), and 0.66% (33.4 μg equiv./cm²) of the applied dose in Test Preparation 3 (ca 100%, w/w).

Executive summary:

The test item, N‑1‑naphthylaniline (PANA) is used as an antioxidant in various oils such as lubricants and in the production of rubber. As part of the safety evaluation of PANA, a study was conducted to assess the absorption following dermal application in accordance with the OECD 428 and under GLP compliance. Exposure to the test item could occur during handling of the pure compound and during processing, for example, after dilution in a base oil for lubricants or after incorporation into a rubber matrix. Concentrations of PANA tested within the scope of this study represented the neat product (ca 100% (w/w) moistened with water); and relevant in-use concentrations diluted in base oil for lubricants (ca 5% (w/v) and ca 1% (w/v)). Due to the use pattern of the test item, a rubber matrix was selected to generate a relevant and technically feasible test sample representing a worst-case sample of the test item using a simplified manufacturing method based on the actual industrial processing (ca 3.5% (w/w) and ca 0.5% (w/w) in a rubber disc).

 

Split‑thickness human skin membranes were mounted into static diffusion cells in vitro. Receptor fluid was added to the receptor chamber (nominal volume 10 mL, nominal exposure area 3.14 cm²). The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment. An electrical resistance barrier integrity assessment was performed and any skin sample exhibiting resistance lower than 4.0 kΩ was excluded from subsequent absorption measurements.

 

The rubber discs and Test Preparation 1-3 were applied to human split‑thickness skin membranes from four to six different donors and the cells were left open to the atmosphere. Test item stability following dosing was confirmed by high performance liquid chromatography (HPLC).

 

Percutaneous absorption was assessed by collecting receptor fluid at 1, 2, 4, 8 and 12 h post dose. At 8 h post dose, the exposure period was terminated by removing the rubber disc (if applicable) then washing the skin surface with a concentrated commercial hand wash soap followed by rinsing with a dilute soap solution (2%, v/v) and drying the surface with tissue paper. At 24 h post dose, the skin was then removed from the static cells, the stratum corneum tape stripped and the skin divided into exposed and unexposed skin. The exposed skin was heat separated into dermis and epidermis samples. The bulk receptor fluid was collected from the receptor chamber and retained for analysis. The donor chambers and receptor chambers were rinsed with ethanol and the samples retained for analysis. The skin samples were dissolved with Solvable™ tissue solubilizer. All samples were analysed by liquid scintillation counting.

Following topical application of [phenyl U 14C]PANA to human skin in vitro, dermal absorption after accounting for variability between replicates is 0.38% (17.6 μg equiv./cm²) of the applied dose in rubber discs (ca 3.5%, w/w), 0.35% (1.91 μg equiv./cm²) of the applied dose in rubber discs (ca 0.5%, w/w), 5.6% (5.84 μg equiv./cm²) of the applied dose in Test Preparation 1 (ca 1%, w/v), 6.4% (34.4 μg equiv./cm²) of the applied dose in Test Preparation 2 (ca 5%, w/v), and 0.66% (33.4 μg equiv./cm²) of the applied dose in Test Preparation 3 (ca 100%, w/w).

Description of key information

Available experimental data on absorption, distribution, metabolism and excretion (ADME) are used to assess the absorption behaviour of N-phenyl-1-naphthylamine and, in the absence of sufficient data, default assumptions are made following the worst case.

Animal data on rats demonstrated that N-phenyl-1-naphthylamine is well absorbed after ingestion and is readily excreted; accumulation in the body is not expected. In vitro studies showed that the metabolism occurs primarily via hydroxylation.

Since the study results suggest that the substance is well absorbed via oral route, it cannot be concluded that the oral absorption is significantly less than 100%. Thus, in accordance with ECHA’s Endpoint specific guidance R.7c (v3.0, June 2017) a default value of complete (i.e., 100%) oral absorption is assumed. 

According to ECHA’s Endpoint specific guidance Chapter R.7c (v3.0, June 2017) a default value of 100% skin absorption is generally used when the molecular mass of the substance is below 500 g/mol and the log P value is in the range of -1 to 4. Also, it is generally assumed that dermal absorption will not be higher than oral absorption.

A skin absorption study following OECD 428 was performed due to the ECHA final decision and the assumption, that dermal absorption of N-phenyl-1-naphthylamine is significantly lower than the default value.
Based on the available dermal absorption study the obtained dermal absorption values are 0.66 % for the neat compound, 0.38 % for 3.5 % in rubber matrix, 0.35 % for 0.5 % in rubber matrix, 5.6 % for 1% in base oil for lubricants and 6.4 % for 5 % in base oil for lubricants. Thus, as key values for the chemical safety assessment dermal absorption is set to 0.66 % for the neat compound (100%), to 5.6 % for lubricant formulations at and below 1%, to 6.4 % for lubricant formulations at and above 5 % and as a worst case to 0.38 % for rubber formulations at 1-5 %.

With regard to inhalation, in the absence of bioavailability data it is most precautionary to assume that 100% of the inhaled vapour is bioavailable. Considering the lack of route-specific information when deriving a “no effect level” for inhalation a default safety factor of 2 is included for route-to-route extrapolation (oral-to-inhalation extrapolation), in line with ECHA's Guidance R.8.

Key value for chemical safety assessment

Bioaccumulation potential:

no bioaccumulation potential

Absorption rate - oral (%):

100

Absorption rate - dermal (%):

0.66

Absorption rate - inhalation (%):

100

Additional information

Absorption following oral exposure

Male Sprague-Dawley rats were orally administered a single dose of 160 mg/kg bw [¹⁴C] N-phenyl-1-naphtylamine. The test material was well absorbed, metabolised almost completely excreted via faeces and urine (Sikka et al., 1981). Radioactivity was detected in plasma within 60 min, with the maximum concentration measured after 4 hours. After 24 hours, 20% of the radioactivity was found in the gastrointestinal tract, 2.4% in fatty tissue, 0.4% in the liver, and 0.1% in the kidneys. More than 90% of the administered dose had been excreted within 48 hours after administration. The primary route of excretion of radioactivity was via the feces (60%), although appreciable amounts of ¹⁴C were also excreted via the urine (32%). In the ether extract of the urine, using HPLC analysis at least five ¹⁴C-metabolites were detected, but no N-phenyl-1-naphthylamine could be determined in the ether extracts. The elimination half-lives were reported as 1.68 hours for the fast elimination and 33 hours for the slow elimination. Mono- and di-hydroxylated derivatives of have been detected in in vitro metabolic studies conducted with rat liver microsomes. Sikka et al. (1981) suggested that the metabolism ofis primarily via hydroxylation and subsequently undergoes with O-glucuronidation or O-sulfation. In the mono-hydroxy derivative, the hydroxy group is in the naphthalene moiety at the position para to the amino group and in the di-hydroxy derivative, at least one hydroxy group is at the available para position in the naphthyl ring. In the in vitro (rat heptic microsomes) metabolism study of Xuanxian and Wolff (1992), only a mono-hydroxylated metabolite was identified. Pretreatment of male rats with phenobarbital or 3-methylcholanthrene increased the rate of microsomal metabolism of, indicating that more than one P-450 monooxygenase mediates the reaction. The formation of 1-naphthylamine via eliminiation of the phenol group of the hydroxylated metabolites is not expected. Based on the chemical structure, it is unlikely that it is metabolised to the carcinogenic 2-naphthylamine.

Absorption following dermal exposure

As part of the safety evaluation of PANA, a study was conducted to assess the absorption following dermal application in accordance with the OECD 428 and under GLP compliance. Exposure to the test item could occur during handling of the pure compound and during processing, for example, after dilution in a base oil for lubricants or after incorporation into a rubber matrix. Concentrations of PANA tested within the scope of this study represented the neat product (ca 100% (w/w) moistened with water); and relevant in-use concentrations diluted in base oil for lubricants (ca 5% (w/v) and ca 1% (w/v)). Due to the use pattern of the test item, a rubber matrix was selected to generate a relevant and technically feasible test sample representing a worst-case sample of the test item using a simplified manufacturing method based on the actual industrial processing (ca 3.5% (w/w) and ca 0.5% (w/w) in a rubber disc).

Split‑thickness human skin membranes were mounted into static diffusion cells in vitro. Receptor fluid was added to the receptor chamber (nominal volume 10 mL, nominal exposure area 3.14 cm²). The skin surface temperature was maintained at 32°C ± 1°C throughout the experiment. An electrical resistance barrier integrity assessment was performed and any skin sample exhibiting resistance lower than 4.0 kΩ was excluded from subsequent absorption measurements.

Following topical application of [phenyl U 14C]PANA to human skin in vitro, dermal absorption after accounting for variability between replicates is 0.38% (17.6 μg equiv./cm²) of the applied dose in rubber discs (ca 3.5%, w/w), 0.35% (1.91 μg equiv./cm²) of the applied dose in rubber discs (ca 0.5%, w/w), 5.6% (5.84 μg equiv./cm²) of the applied dose in Test Preparation 1 (ca 1%, w/v), 6.4% (34.4 μg equiv./cm²) of the applied dose in Test Preparation 2 (ca 5%, w/v), and 0.66% (33.4 μg equiv./cm²) of the applied dose in Test Preparation 3 (ca 100%, w/w).